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The postnatal ontogeny of gonadotroph cells in the female sheep
Journal of Chemical Neuroanatomy 31 (2006) 130–138 www.elsevier.com/locate/jchemneu
The postnatal ontogeny of gonadotroph cells in the female sheep Developmental patterns of synthesis, storage and release of gonadotrophic hormones Marta Wan´kowska *, Jolanta Polkowska Department of Endocrinology, The Kielanowski Institute of Animal Physiology and Nutrition, Polish Academy of Sciences, 05-110 Jabłonna, Poland Received 7 July 2005; received in revised form 3 October 2005; accepted 4 October 2005 Available online 14 November 2005
Abstract The aim of this study was to determine the developmental changes in the synthesis, storage and release of luteinizing hormone (LH) and folliclestimulating hormone (FSH) in the hypophyseal gonadotroph cells from infancy to peripuberty of ovine ontogeny. An examination has been made in 15 infantile (12-, 15-week-old) and juvenile (22-, 30-week-old) ovary-intact sheep. Histomorphological and functional changes in the adenohypophyseal population of gonadotrophs were assayed with hybridohistochemistry, immunohistochemistry and radioimmunoassay. The percentage of the adenohypophyseal area (PAA) occupied by gonadotrophs containing LHb-mRNA or FSHb-mRNA was highest (P < 0.05) in the 15-week-old sheep compared with the other stages. The gradual increase in the PAA occupied by immunoreactive (ir)-LHb-cells from the 12th to 30th week of age was observed (P < 0.05) and has been interpreted as the increase in the storage of LH. This histomorphological change was accompanied by the gradual increase in the LH pulse frequency from the 15th to 30th week of age (P < 0.05). The PAA occupied by ir-FSHb-cells was extremely high in the infantile sheep, and subsequently, low in the juvenile sheep (P < 0.05). Altogether, similar patterns of pretranslational synthesis of the LHb- and FSHb-subunit but clearly different storage patterns of gonadotrophins were observed. The postnatal development of gonadotrophins seems to be determined by the progressive increase in the storage of LH until peripuberty and by the acute decrease in the storage of FSH during the infantile/juvenile shift. These findings imply the different intrahypophyseal regulation of LH and FSH posttranscriptional processing during the period of transition between infancy and peripuberty in female sheep. # 2005 Elsevier B.V. All rights reserved. Keywords: Sexual development; Gonadotrophic hormones Hybridohistochemistry; Immunohistochemistry; Radioimmunoassay; Female lamb
1. Introduction Sexual development depends on the precise and coordinated functions of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). The adenohypophyseal gonadotroph population, as the site of synthesis, storage and release of LH and FSH, is the morphological basis of the effector mechanism of neuroendocrine functions of the reproductive system (Meeran et al., 2003). The development of the gonadotrophic axis from birth to the peripubertal period has been poorly described in sheep. Very little is known about the postnatal changes of ovine gonadotroph cells’ endocrine functions except the developmental patterns of circulating LH and FSH. These patterns can reflect functional changes of the gonadotrophic axis on the
* Corresponding author. Tel.: +48 22 7824037; fax: +48 22 7742038. E-mail address: [email protected] (M. Wan´kowska). 0891-0618/$ – see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jchemneu.2005.10.002
systemic level and are described by Foster et al. (1975a,b) and Huffman et al. (1987). To our knowledge, the only study related to postnatal changes in the gonadotroph population revealed that in newborn female lambs the percentages of LH and LH– FSH cells do not change, whereas FSH cells percentage increases about two-fold after birth (Messaoud-Toumi et al., 1993). The intrahypophyseal mechanisms underlying the developmental changes in gonadotrophins on the systemic level during the period of transition between infancy and puberty in sheep are unknown. The present work was designed to describe the change from infantile to peripubertal activity of LH and FSH in the adenohypophyseal gonadotroph population of the ovary-intact sheep. The histomorphological developmental changes in the storage and/or synthesis patterns of gonadotrophins in the adenohypophysis were demonstrated by their immunoreactive (ir) or in situ hybridised contents. The immunoreactivity for adenohypophyseal LHb, FSHb and in situ hybridised LHb-
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mRNA or FSHb-mRNA were investigated by immunohistochemical or hybridohistochemical techniques. Additionally, the release of LH into the peripheral circulation was determined by radioimmunoassay. The comparison of adenohypophyseal populations of cells synthesising or storing LH or FSH between postnatal stages of ovine ontogeny may help to describe the nature of the effector mechanism of sexual neuroendocrine development of sheep. 2. Materials and methods 2.1. Animals and tissue preparation The study was performed on purebred Polish Merino female sheep born in the first week of July on the Bieganowo Farm (Poland). Thus, the lambs were born after the summer solstice, i.e. out of season. Lambs were housed in an outdoor environment and left to graze with ewes on pasture for 12 weeks after parturition. Twelve-week-old ovary-intact females were divided randomly into four age groups according to different phases of postnatal ontogenesis— infantile and juvenile periods. Infancy was represented by 12-week-old unweaned sheep (average weight 18.3 1.17 kg; n = 3) and 15-week-old weaned lambs (2 weeks after weaning; average weight 21.1 2.16 kg; n = 4). The juvenile period (adolescence) was represented by 22-week-old prepubertal lambs (2 months around the expected time of puberty; average weight 25.9 2.95 kg; n = 4) and 30-week-old peripubertal lambs (2 weeks around the expected time of puberty; average weight 32.3 2.80 kg; n = 4). The 12-week-old unweaned lambs (sucked their dams one to two times per 24 h) were independent of their mothers in respect of feeding and grazed with flock in the pasture. The slaughter of these lambs was conducted on the local licensed slaughterhouse of the Bieganowo Farm. All the other lambs were transported from the farm to the Institute of Animal Physiology and Nutrition (Jabłonna, Poland) shortly after their weaning at 13 weeks of age. Lambs were maintained indoors in double pens under natural lighting and temperature conditions at 528N and 218E. Animals were fed a diet of commercial concetrates, with hay and water available ad libitum. Every 3 days over a period of 29–30 weeks of age, three consecutive blood samples were taken for determination of progesterone concentrations in the peripheral blood plasma. One day before each experiment day (from 15th to 30th week of age), a jugular venous catheter was inserted and kept with heparinised saline (50 units of heparin/ml in 0.9%, w/v, NaCl). On experiment days, blood samples were taken at 10 min intervals over a 6 h period. Samples were centrifuged and the collected plasma was stored at 20 8C until analysis. During sample collection, the animals were kept in comfortable cages, where they could lie down and have unrestrained access to hay and water. Shortly after the collection, lambs were anesthetised with an i.v. injection of pentobarbitone sodium (20 mg/kg; Biochemie GmbH, Kundl, Austria) and were slaughtered by decapitation in the local licensed slaughterhouse. The ovaries of the 30-week-old lambs, were inspected after slaughter. All procedures were approved by the Local Ethics Committee in Warsaw, according to the Polish Law for the Care and Use of Animals (2 August 1997). Immediately after decapitation, brains were perfused with 1000 ml 0.1 M phosphate buffered saline (PBS; Sigma, St. Louis, USA) and subsequently with 1500 ml 0.1 M PBS containing 4% (w/v) paraformaldehyde (Sigma–Aldrich, Seelze, Germany) and 15% saturated picric acid (MERCK, Darmstadt, Germany) solution (w/v), pH 7.4, via both carotid arteries. The hypophyses were dissected 20 min after the beginning of perfusion and postfixed for 48 h by immersion in the same fixative and washed with 0.01 M PBS. All solutions used for the following steps of tissue preparation and fixation were made up fresh and sterile autoclaved. The hypophyses were dehydrated in graded alcohol, embedded in paraplast (Sigma, St. Louis, USA) and 4 mm sections were cut in the sagittal plane.
2.2. Preparation of probes LHb- and FSHb-sense and antisense riboprobes were produced using previously obtained homologous sheep double-stranded cDNAs (D’Angelo-Bernard
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et al., 1990; Pelletier et al., 1992, 1995). cDNAs (533 bp oLHb-cDNA or 900 bp oFSHb-cDNA) were digoxigenin (DIG) labelled by in vitro transcription with SP6 and T7 RNA polymerase using the DIG-labelling kit from Roche Molecular Biochemicals (Meylan, France) and pGEM-TEasy vectors (Promega, Charbonnieres, France). The specificity of probes was confirmed by the absence of a positive signal in sections hybridised with sense probes (data not shown).
2.3. Hybridohistochemistry The cellular LHb-mRNA and FSHb-mRNA was studied by an in situ hybridisation technique in combination with an IHC detection of DIG. Sections were mounted on slides and coated with 3-aminopropyltriethoxysilane (2% in acetone; Sigma, St. Louis, USA). All sections from every stage were run in a single assay. The non-radioactive in situ hybridisation procedure using DIG-labelled cDNA probes was performed according to Breitschopf et al. (1992) protocol, with minor modifications by Wan´kowska et al. (2002). Briefly, slides were deparaffined, rehydrated, washed in PBS, fixed with 4% paraformaldehyde in PBS and treated with 0.25% acetic anhydride in 100 mM TEA buffer (triethanolamine-Cl; Sigma, St. Louis, USA). Next, the sections were treated with 200 mM HCl, then with 1% proteinase K (ICN, Aurora, OH, USA) dissolved in Tris–EDTA and finally washed in Tris–glycine buffer. Sections were prehybridised at 55 8C in a humid chamber and in situ hybridisation was carried out using DIG-dUTP antisense or sense RNA probes diluted 1:100. After incubation, slides were washed in 2 SSC and treated with 20 mg/ml RNase A (Sigma, St. Louis, USA) in 0.5 M NaCl, 10 mM Tris–HCl (pH 8.0), 10 mM EDTA. Next, the sections were treated with 30% formamide (Sigma, St. Louis, USA) and washed in 2 SSC. The sections were washed in 100 mM Tris–HCl (pH 7.5), 150 mM NaCl, 0.5% blocking reagent (Boehringer, Mannheim, Germany). Next, the sections were incubated with alkaline phosphatase anti-DIG antibody (Boehringer, Mannheim, Germany) diluted 1:500 in the same buffer. Following this, the sections were washed in 100 mM Tris–NaCl (pH 7.5), 150 mM NaCl for 15 min and for 3 min in 100 mM Tris–HCl (pH 9.5), 100 mM NaCl and 50 mM MgCl2. After that the sections were incubated in the last buffer containing 75 mg/ml NBT, 50 mg/ml BCIP (Boehringer, Mannheim, Germany) and 10 mM levamisole (Sigma, St. Louis, USA). The colour reaction was stopped in water and slides were mounted in glycerol-gelatine (Sigma, St. Louis, USA). Controls were prepared using RNase pretreatment. Sections were incubated with RNase (Sigma, St. Louis, USA), 1 unit/100 ml 10 mM Tris–HCl buffer, pH 7.9, per section, just prior to the addition of the hybridisation buffer for 30 min, and then, slides were washed with 4 SSC. Controls did not exhibit any staining (data not shown).
2.4. Immunohistochemistry (IHC) The populations of LH- or FSH-containing cells in the adenohypophysis were determined by means of immunohistochemical light microscopy. All sections taken from all animals were run in a single assay. Hypophyseal sections were deparaffined, rehydrated, washed in 0.01 M PBS, incubated for 30 min in 2% preimmune normal lamb serum in 0.01 M PBS and 30 min in 0.1% hydrogen peroxide (Chempur, Piekary S´la˛skie, Poland) in 0.01 M PBS. Sections were incubated with primary antisera raised in the rabbit: anti-ovine LHb No. I3 diluted 1:1000 and anti-ovine FSHb No. P5 diluted 1:200 for 4 days at 4 8C. Methodological details of their preparation and their specificity were described by Hurbain-Kosmath et al. (1990). After the incubation with primary antibodies, sections were rinsed in 0.01 M PBS, incubated for 2 h at room temperature with the secondary antibody (sheep anti-rabbit Ig [H + L] labelled with peroxidase; Bio-Rad, Steenvoorde, France) diluted 1:40 in 0.1% normal lamb serum in 0.01 M PBS. The colour reaction was developed by incubating sections with 0.05% 30 ,3-diaminobenzidine tetrahydrochloride chromogen (Sigma, St. Louis, USA) and 0.001% hydrogen peroxide in 0.05 M Tris buffer (ICN, Aurora, OH, USA). For control staining, the primary antiserum was replaced with the same dilution of rabbit serum. As an additional control reaction, the inhibition of antihormone serum with its homologous antigen was used. To evaluate the specificity of staining of LH and FSH, corresponding antisera were preadsorbed using synthetic rLH32V02 and rFSH32V02 (National Hormone and Pituitary Program, Torrance, CA, USA). Synthetic antigens and antisera were mixed and
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preincubated for 24 h at 4 8C and then used for the IHC staining instead of the primary antibody. Neither of the controls exhibited any specific staining (data not shown).
by the elaborated small and comparable movements (16 for each section) of screw moving the slide on the microscope stage. The frame size was kept constant for the duration of the image analysis.
2.5. Morphometric analysis
2.6. Determination of LH and progesterone in peripheral blood plasma
A type 104 Nikon projection microscope (Nikon Corporation, Yokohama, Japan) was used for morphometric analyses of hypophyseal sections. The staining was analysed using the ‘‘Lucia’’ image analysis computer system Version 3.51ab (Laboratory Imaging Ltd., Prague, Czech Republic). Analyses were performed under a 40 objective. Pictures of stained sections were projected by a camera (Panasonic KR222, Matsushita Electric Industrial Co., Osaka, Japan) to a colour monitor. Images were adjusted for optimal contrast, fixed at the same brightness level, converted to grey and processed by background subtraction and removal of artifacts. The area fraction parameter defining the percentage of the total area of adenohypophyseal pars distalis that exhibited specific staining was measured. This parameter expresses pool of positively stained cross-sections of gonadotrophs in the total adenohypophyseal cell number. Quantitative analyses were performed for each hypophysis in subareas of pars distalis using a mechanical threshold function to select a range of grey values that were optically identified as positive staining. The analyses of the immunoreaction and/or in situ hybridisation signal in gonadotrophs were made in four sections of each adenohypophysis, using every 40th section (16 fields of 0.0837 mm2 measured in each section). Regions of interest in the pars distalis (for every jointly 64 measured fields) were random—chosen for analysis
The concentration of LH was determined in duplicate 100 ml aliquots by a routine double-antibody radioimmunoassay (RIA) procedure using anti-ovine LH, anti-rabbit-gammaglobuline antisera and bovine LH standard NIH-LH-B6 according to Stupnicki and Madej (1976). The assay detection limit was 0.06 ng/ml sample. The coefficient of variation calculated for control samples at concentration 1 and 5 ng/ml of LH was 10 and 4%, respectively. The concentration of FSH could not be measured due to technical problems. The concentration of progesterone was assayed in duplicate 100 ml aliquots by a direct RIA method routinely used in our laboratory according to procedure described by Stupnicki and Kula (1982) with a sensitivity of 6.2 pg/ml. The intra-assay coefficient of variation was <10%.
2.7. Statistical analyses The quantitative measurements taken from each section for each adenohypophysis were averaged to obtain a mean estimate for each adenohypophysis for each animal. Then, the mean data were pooled to represent the individual age groups. Thus, the data are reported as the mean percentage (for 12-week-old
Fig. 1. Adenohypophyseal gonadotrophs containing in situ hybridised mRNA transcripts encoding LHb (a and b) or FSHb (c and d) subunits in the representative 15week-old lamb (the infantile period; a and c) and 30-week-old lamb (the juvenile period; b and d). Note the higher populations of LHb-mRNA-positive or FSHbmRNA-positive cells in infantile sheep compared with juvenile sheep. Calibration bars 50 mm.
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sheep, n = 3, for other n = 4 per stage) S.E.M. of the total area that exhibited positive and specific immunostaining or hybridohistochemical staining according to developmental stages. Significant effects of postnatal age on the factors studied were assessed by one-way analysis of variance (ANOVA) followed by the post hoc Tukey’s test to determine significant differences between means for individual age groups, using the Statistica 6.0 PL software (StatSoft Inc., Tulsa, OK, USA). Data of RIA are presented as mean (n = 4 per stage) S.D. The mean concentration of LH for individual animals was calculated from the area under the curve (the sum of trapezoid areas between the curve and the abscissa). Pulse characteristic of LH were calculated using the Pulsar Computer Program Version 3.01 developed by Merriam and Wachter (1982) and adapted to operate on an IBM-PC. The cut-off parameters G(n) were settled to a 5% error rate assuming a normal distribution of data. The level of significance for differences between stage groups for data obtained from plasma LH analysis was calculated using the one-way analysis of variance, followed by the post hoc Newman– Keuls test (for mean concentration and amplitude of pulse) or the Kruskal– Wallis test, the non-parametric equivalent of ANOVA for number of pulses. Significance was defined at the P < 0.05 level.
3. Results 3.1. Animals The progesterone concentrations in blood plasma did not exceed 0.5 ng/ml in sheep. Inspection of the 30-week-old sheep ovaries revealed no corpora lutea and at least one dominant ovarian follicle of about 6–7 mm in diameter. 3.2. In situ hybridised LHb-mRNA and FSHb-mRNA No detectable hybridohistochemical signal was observed in sections treated with sense probes for LHb-mRNA or FSHbmRNA (results not shown). Microscopic analysis of the hybridohistochemically stained gonadotrophs showed that within the pituitaries of lambs between 12th and 30th week of age the populations of LHb-mRNA- and FSHb-mRNApositive cells were highest in the 15-week-old sheep compared with the other stages of postnatal development (Fig. 1). Microscopic observations were supported by quantitative computer estimation of the percentages of the adenohypophyseal area occupied by gonadotrophs containing hybridised LHb-mRNA or FSHb-mRNA, which were highest (P < 0.05) in the 15-week-old sheep in comparison with the other investigated stages (Fig. 2). 3.3. Immunoreactive gonadotrophins The population of adenohypophyseal cells immunostained positively for LH increased gradually with progressive developmental stages, up to the expected time of the peripubertal period (Fig. 3). This was reflected by the significant gradual increase in the percentage of adenohypophyseal area occupied by ir-LHb-positive cells within the pituitaries of lambs between the 12th and 30th week of age (from 2.45 0.064 to 5.42 0.140%; P < 0.05; Fig. 4). In contrast to ir-LHb-positive cells, the pool of adenohypophyseal cells immunostained positively for FSH was the highest during the 12th and 15th weeks of age and the lowest during the 22nd and 30th weeks of age (Fig. 3). The percentage of
Fig. 2. The percentage of adenohypophyseal area occupied by gonadotrophs containing in situ hybridised LHb-mRNA (a) or FSHb-mRNA (b) within the pituitaries of lambs between the 12th and 30th week of age. Values are mean S.E.M.; *P < 0.05. Note the similar developmental patterns of the pretranslational synthesis of the both LHb- and FSHb-subunit.
adenohypophyseal area occupied by ir-FSHb-cells during the investigated developmental period was higher (P < 0.05) and relatively stable in the 12- and 15-week-old sheep (3.41 0.088 and 3.45 0.159%; Fig. 4) and subsequently, lower (P < 0.05) and stable in the 22- and 30-week-old sheep (1.26 0.068 and 1.25 0.068%; Fig. 4). 3.4. LH in peripheral blood plasma The LH concentration was higher (P < 0.05) in the 30week-old sheep compared with the earlier stages of postnatal
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Fig. 3. Adenohypophyseal gonadotrophs containing immunoreactive LHb (a and b) or FSHb (c and d) in the representative 12-week-old lamb (the infantile period; a and c) and 30-week-old lamb (the juvenile period; b and d). Note the lower population of LH-containing gonadotrophs and the higher population of FSH-containing gonadotrophs in infantile lamb in comparison with juvenile lamb. Calibration bars 50 mm.
development (Fig. 5a). A gradual increase (P < 0.05) in the LH pulse frequency from the 15th to 30th week of age was observed (from 3 1 to 8 1, number of LH pulses/6 h; P < 0.05; Fig. 5b). No differences were observed in LH pulse amplitude (Fig. 5c). 4. Discussion The subsequent developmental changes in the adenohypophyseal cells producing LH and/or FSH are analysed and compared with hybridohistochemistry, immunohistochemistry and radioimmunoassay. The populations of stained gonadotrophs were higher or lower according to the level of synthesising or releasing cellular activity present until the moment of the tissue’s fixation after decapitation of lambs. 4.1. Developmental patterns of synthesis and storage of LH and FSH The change of lower population of gonadotrophs stained by hybridohistochemistry into higher one has been interpreted as the increase in the gonadotrophic b subunits
transcriptional synthesis and/or stability of mRNAs. The regulation of gonadotrophin subunits’ gene expression and translation may be influenced by alterations in stability and half-life of mRNA (see for review, Crawford and McNeilly, 2002). The pretranslational synthesis of b subunits of LH and FSH in the gonadotroph population was highest during the shift from the infantile to the juvenile period compared with the other stages of postnatal development. Similar developmental changes of gonadotrophin b subunit mRNAs were assessed in the female rat (Zapatero-Caballero et al., 2004). Namely, LHb-mRNA and FSHb-mRNA levels as determined by Northern blot analysis rose slowly during the infantile period and thereafter, the levels of both mRNAs fell during the juvenile period until peripuberty (Zapatero-Caballero et al., 2004). Thus, similar developmental patterns of pretranslational synthesis of the LHb- and FSHb-subunit were observed during the postnatal development of female sheep. These pretranslational patterns were accompanied by differential posttranslational regulation of LH and FSH storage. The change of lower population of adenohypophyseal gonadotrophs stained by IHC into higher one has been
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Fig. 4. The percentage of adenohypophyseal area occupied by gonadotrophs containing immunoreactive LHb (a) or FSHb (b) subunit within pituitaries of lambs between the 12th and 30th week of age. Values are mean S.E.M.; * P < 0.05. Note the clearly different storage patterns of gonadotrophins according to investigated stages of ontogeny.
interpreted as the increase in the storage, whereas the opposite change has been interpreted as the decrease in the accumulation of hormone. The storage of LH increased gradually until the expected time of the peripubertal period, whereas the storage of FSH was extremely high during infancy and extremely low during the juvenile period. Thus, clearly different storage patterns of LH and FSH were observed during the period of transition between infancy and peripuberty in the female sheep. Previously, dynamic postnatal modifications of the heterogeneity of LH and FSH storage were observed in the
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hypophysis of the female rat (Jansen, 1982). The numbers of gonadotrophs per pituitary tissue and the amounts of LH and FSH in pituitary cells increased during the infantile period. Then, the number of LH cells remained high until puberty, whereas the number of FSH cells decreased (Jansen, 1982). Furthermore, in the male rhesus monkey, the pituitary gonadotroph population increased, and a large number of monohormonal FSH gonadotrophs were likely to become bihormonal during the juvenile–adult transition period (Meeran et al., 2003). Meeran et al. (2003) suggested that these findings attest that gonadotrophic cells can be multipotent, switching their hormonal identity in systemic response to different environmental influences. Altogether, similar patterns of the LHb- and FSHb-subunit pretranslational synthesis, but clearly different storage patterns of gonadotrophins were observed in the postnatally developing female sheep. The results obtained in the current investigation on sheep support the discussed above observations related to the rat and monkey (Jansen, 1982; Meeran et al., 2003; ZapateroCaballero et al., 2004). This similarity led us to define and employ of the new one division in the postnatal sexual development of sheep, on the basis of patterns of the both gonadotrophins’ synthesis and the specific storage of FSH. As in the rat and monkey, the ovine postnatal sexual development is divided now into: (i) the infantile period of increase in the both gonadotrophins’ synthesis and the high storage of FSH (after the neonatal period, to the transitional stage beyond the time of weaning jointly) and (ii) the juvenile period of decrease in the both gonadotrophins’ synthesis and low storage of FSH (beginning no earlier than after the time of weaning and represented by prepuberty and peripuberty). The terminology and duration of the different stages of postnatal sexual development of sheep have been defined firstly by Foster et al. (1975a) on the basis of patterns of circulating LH. The postnatal secretion of LH in the female sheep was often pulsatile and by 11 weeks of age serum LH attained levels far above adult baseline. Further, during sexual maturation and early adulthood concentrations of circulating LH and its baseline levels remained stable and greater than those in the mature cycling ewe (Foster et al., 1975a,b). Thus, the tonic LH secretion during the course of postnatal ontogeny of female sheep has been divided by Foster et al. (1975a) into the following three phases: (i) a neonatal period (the first 2–5 weeks) of low secretion; (ii) a period of high secretion during sexual maturation and early adulthood; (iii) a period of low secretion during true adulthood. However, these patterns can reflect functional changes of the gonadotrophic axis only on the systemic level. Thus, in the current study, the new one division in the postnatal sexual development of female sheep has been defined on the basis of intrapituitary changes in the synthesis and storage of both gonadotrophins. The observations obtained in this investigation led to the hypothesis that regulatory mechanisms responsible for divergent LH and FSH secretion during the postnatal development of female sheep may be explained by the differential posttranscriptional regulation of both gonadotrophins.
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Fig. 5. The plasma LH concentration (a), frequency (b) and amplitude (c) in ovary-intact female lambs during transition from the infantile to the peripubertal period of ontogeny. Values are mean (for every n = 4) S.D.; *P < 0.05. Note the higher plasma LH concentration in the 30-week-old sheep, the gradual increase in the LH pulse frequency from the 15th to 30th week of age and no differences in the LH pulse amplitude according to investigated stages of ontogeny.
4.2. Regulatory mechanisms of divergent posttranscriptional processing of LH and FSH In the current study, the plasma LH concentration was stable during the shift from infancy to the juvenile period and during prepuberty. Then, it was enhanced at the expected time of peripuberty. Furthermore, the gradual increase in LH pulse frequency accompanied by stable pulse amplitude during the transition period between infancy and the expected time of the peripubertal period was observed. It is generally accepted that an increase in the frequency of episodic LH secretion is a key event leading to the onset of ovarian cycles in the lamb, whereas an increase in pulse amplitude must occur just before the LH surge (Huffman et al., 1987). The lambs from the present investigation were born out of season, i.e. after the summer solstice, what differs from most of the literature published on the topic. However, the current results of pulse frequency and concentration of LH in the ovary-intact Merino lambs, which are a less photoperiodic breed, can be compared to findings obtained from ovariectomised highly seasonal Suffolk lambs, born near the spring equinox and maintained in a permissive sequence of photoperiods in the presence of estradiol replacement (Ebling et al., 1990). The current findings describing regulatory mechanisms of divergent posttranscriptional processing of LH and FSH cannot be compared to results related to sheep born in spring, due to the fact that there are not any results related to the synthesis or storage of both gonadotrophins obtained from postnatally developing female lambs born near the spring equinox. It should be pointed out that the current study simply proposes intrahypophyseal mechanisms, which might account for the postnatal development of the ovine gonadotrophic axis. In this study, during the juvenile period, the decrease in the adenohypophyseal population of cells containing LHb-mRNA accompanied by the increase in the LH-containing cell
population and in the plasma LH pulse frequency were observed. Thus, we hypothesize that changes in the histological feature of gonadotroph population, dependent on the increase in the number of LH-containing cells, can represent the intrahypophyseal posttranscriptional mechanisms that differentially regulate the secretion of both gonadotrophins during the transition period between infancy and adulthood in the female sheep. Furthermore, the postnatal development of gonadotrophins seems to be determined by the acute and characteristic decrease in the storage of FSH during the ovine infantile/juvenile transitional stage. This is in agreement with a suggestion of Meeran et al. (2003), that, the secretion of both LH and FSH throughout sexual development could be differentially regulated by changes in the proportion of gonadotroph subtypes within the total pituitary gonadotroph population. Moreover, Crawford and McNeilly (2002) suggested that in the female sheep, the majority of synthesised LH seems to be laid aside into storage (McNeilly et al., 1991) and to be released upon extracellular stimulation, directly mimicking the pulsatile secretion of gonadotrophin-releasing hormone (GnRH) (Clarke and Cummins, 1982). Thus, a characteristic of the GnRH/LH secretory pathway is the ability of LH protein to be stored (Crawford and McNeilly, 2002). Crawford and McNeilly (2002) revealed that the secretory pattern of LH does not reflect its transcriptional rate, but is related to posttranslational intrapituitary mechanisms due to the storage and transportation of secretory granules within the gonadotrophs of the ewe (Thomas and Clarke, 1997). This intracellular mechanism has been suggested by Currie and McNeilly (1995) to switch non-releasing gonadotrophs into a potentially releasable state. Some LH is additionally released constitutively (McNeilly et al., 1991), possibly to maintain basal levels of LH (Crawford et al., 2000). In view of discussed above discrepancies, the findings of the current study imply the existence of the histomorphological feature of the intrahypo-
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physeal regulation of posttranscriptional processing and release of LH during the transition from infantile to the peripubertal activity of gonadotroph population in the female sheep. The data obtained in the present investigation reveal the increase in the population of cells containing FSHb-mRNA accompanied by the extremely high number of FSH-containing cells during the infantile period. During the juvenile period, the decrease in the population of cells containing FSHb-mRNA accompanied by the extremely low number of FSH-containing cells was observed. Concentrations of FSH in the blood plasma were not determined due to technical problems, but it is perhaps pertinent to notice that Foster et al. (1975b) made a note of increase in the plasma FSH between the 15th and 20th week of postnatal age. Thereafter, the decrease in the plasma FSH between the 20th and 30th week of postnatal age was observed in ovary-intact female lambs (Foster et al., 1975b). Crawford and McNeilly (2002) suggested that in contrast to the secretion of LH, the release of FSH is primarily constitutive (Farnworth et al., 1988, 1995), i.e. FSH is trafficked to the site of exocytosis shortly after synthesis and is released. Thus, there exists a strong relationship between the level of pituitary FSHb-mRNA and plasma FSH, which is representative for constitutively secreted hormones (Crawford and McNeilly, 2002). In conclusion, the findings of current study imply the existence of the relationship between the pretranslational synthesis and storage of FSH in contrast to the relationship between the storage and pulse frequency of LH during the postnatal sexual development of female sheep. Altogether, the data obtained in the present study and these discussed above, led to the conclusion that it is more probable that the differential regulation of both gonadotrophins in the postnatally developing female sheep is on the level of posttranscriptional processing and release rather than on the level of gene expression. The suggested requirement for sexual maturation is the change in the histomorphological feature of gonadotroph population, dependent on the increase in the number of LH-containing cells. 4.3. Conclusions Similar patterns of pretranslational synthesis of the LHband FSHb-subunits but clearly different storage patterns of both gonadotrophins were observed in the postnatally developing female sheep. These observations lead to the suggestion that the regulatory mechanism responsible for divergent LH and FSH secretion during the postnatal development of female sheep could be explained by the more probable different regulation on the level of storage and release rather, than on the level of gene expression. It is suggested that the histomorphological changes dependent upon: (i) the gradual increase in the storage of LH according to progressive developmental stages and (ii) the acute decrease in the storage of FSH during the infantile/juvenile shift, could represent the intrahypophyseal posttranscriptional mechanisms that differentially regulates the both gonadotrophins’ secretion during the period of transition between infancy and puberty in the female sheep. Altogether, mechanisms of postnatal sexual development in the female sheep, that are
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dependent upon the coordinated function of LH and FSH, are likely to operate within the adenohypophysis and could have at least partly the histomorphological feature. The findings of this study are novel for ruminants and support observations in the rat and monkey. Acknowledgments The authors are grateful to the Mrs. Ewa Skrzeczyn´ska and Mrs. Anna Misztal for technical assistance. They also express their thanks to Dr. Raymond Counis for kind provision with LHb- and FSHb-sense and antisense probes. This study was supported by the Polish Committee for Scientific Research, Grant No. 6 PO6D 008 20. References Breitschopf, H., Suchanek, G., Gould, R.M., Colman, D.R., Lassman, H., 1992. In situ hybridization with digoxigenin-labelled probes: sensitive and reliable detection method applied to myelinating rat brain. Acta Neuropathol. 84, 581–587. Clarke, I.J., Cummins, J.T., 1982. The temporal relationship between gonadotropin releasing hormone (GnRH) and luteinizing hormone (LH) secretion in ovariectomized ewes. Endocrinology 111, 1737–1739. Crawford, J.L., McNeilly, A.S., 2002. Co-localisation of gonadotrophins and granins in gonadotrophs at different stages of the oestrous cycle in sheep. J. Endocrinol. 174, 179–194. Crawford, J.L., Currie, R.J.W., McNeilly, A.S., 2000. Replenishment of LH stores of gonadotrophs in relation to expression, synthesis and secretion of LH after the preovulatory phase of the sheep oestrus cycle. J. Endocrinol. 167, 453–463. Currie, R.J.W., McNeilly, A.S., 1995. Mobilization of LH secretory granules in gonadotrophs in relation to gene expression, synthesis and secretion of LH during the preovulatory phase of the sheep oestrous cycle. J. Endocrinol. 147, 259–270. D’Angelo-Bernard, G., Moumni, M., Jutisz, M., Counis, R., 1990. Cloning and sequence analysis of the cDNA for the precursor of the beta subunit of ovine luteinizing hormone. Nucleic Acid Res. 18, 2175. Ebling, F.J.P., Kushler, R.H., Foster, D.L., 1990. Pulsatile LH secretion during sexual maturation in the female sheep: photoperiodic regulation in the presence and absence of ovarian steroid feedback as determined in the same individual. Neuroendocrinology 52, 229–237. Farnworth, P.G., Robertson, D.M., de Kretser, D.M., Burger, H.G., 1988. Effects of 31 kilodalton bovine inhibin on follicle-stimulating hormone and luteinizing hormone in rat pituitary cells in vitro: actions under basal conditions. Endocrinology 122, 207–213. Farnworth, P.G., Thean, E., Robertson, D.M., 1995. Ovine anterior pituitary production of follistatin in vitro. Endocrinology 136, 4397–4406. Foster, D.L., Jaffe, R.B., Niswender, G.D., 1975a. Sequential patterns of circulating LH and FSH in female sheep during the early postnatal period: effect of gonadectomy. Endocrinology 96, 15–21. Foster, D.L., Lemons, J.A., Jaffe, R.B., Niswender, G.D., 1975b. Sequential patterns of circulating luteinizing hormone and follicle-stimulating hormone in female sheep from early postnatal life through the first estrous cycles. Endocrinology 97, 985–994. Huffman, L.J., Inskeep, E.K., Goodman, R.L., 1987. Changes in episodic luteinizing hormone secretion leading to puberty in the lamb. Biol. Reprod. 37, 755–761. Hurbain-Kosmath, I., Berault, A., Noel, N., Polkowska, J., Bohin, A., Jutisz, M., Leiter, E.H., Beamer, W.G., Bedigian, H.G., Davisson, M.T., Harrison, D.E., 1990. Gonadotropes in a novel rat pituitary tumor cell line, RC-4B/C. Establishment and partial characterisation of the cell line. In Vitro Cell. Dev. Biol. 26, 431–440. Jansen, H.G., 1982. Distribution and number of gonadotrophic cells in the pituitary gland of prepubertal female rats. J. Endocrinol. 94, 381–388.
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McNeilly, J.R., Brown, P., Clark, A.J., McNeilly, A.S., 1991. Gonadotropinreleasing hormone modulation of gonadotrophins in the ewe: evidence for differential effects on gene expression and hormone secretion. J. Mol. Endocrinol. 7, 35–43. Meeran, D., Urbanski, H.F., Gregory, S.J., Townsend, J., Tortonese, D.J., 2003. Developmental changes in the hormonal identity of gonadotroph cells in the rhesus monkey pituitary gland. J. Clin. Endocrinol. Metab. 88, 2934–2942. Merriam, G.R., Wachter, K.W., 1982. Algorithms for the study of episodic hormone secretion. Am. J. Physiol. 243, E310–E318. Messaoud-Toumi, L.H., Taragnat, C., Durand, P., 1993. Heterogeneity in the storage of gonadotropins in the ovine fetus and evidence for luteinizing hormone–follicle stimulating hormone cells in the fetal pituitary. Biol. Reprod. 48, 1239–1245. Pelletier, J., Counis, R., de Reviers, M.M., Tillet, Y., 1992. Localization of luteinizing hormone b-mRNA by in situ hybridization in the sheep pars tuberalis. Cell Tissue Res. 267, 301–306. Pelletier, J., Counis, R., de Reviers, M.-M., Moumni, M., Tillet, Y., 1995. Changes in LHb-gene and FSHb-gene expression in the ram pars
tuberalis according to season and castration. Cell Tissue Res. 281, 127– 133. Stupnicki, R., Kula, E., 1982. Direct radioimmunoassay of progesterone in human plasma. Endokrinologie 80, 1–7. Stupnicki, R., Madej, A., 1976. Radioimmunoassay of LH in the blood plasma of farm animals. Endokrinologie 68, 6–13. Thomas, S.G., Clarke, I.J., 1997. The positive feedback action of estrogen mobilizes LH-containing, but not FSH-containing secretory granules in ovine gonadotropes. Endocrinology 138, 1350–1374. Wan´kowska, M., Lerrant, Y., Wojcik-Gladysz, A., Starzec, A., Counis, R., Polkowska, J., 2002. Intracerebroventricular infusion of neuropeptide Y upregulates synthesis and accumulation of luteinizing hormone but not follicle stimulating hormone in the pituitary cells of prepubertal female lambs. J. Chem. Neuroanat. 23, 133–142. Zapatero-Caballero, H., Sanchez-Franco, F., Fernandez-Mendez, C., GarciaSan Frutos, M., Botella-Cubells, L.M., Fernandez-Vazquez, G., 2004. Gonadotropin-releasing hormone receptor gene expression during pubertal development of female rats. Biol. Reprod. 70, 348–355.
The postnatal ontogeny of gonadotroph cells in the female sheep Developmental patterns of synthesis, storage and release of gonadotrophic hormones Marta Wan´kowska *, Jolanta Polkowska Department of Endocrinology, The Kielanowski Institute of Animal Physiology and Nutrition, Polish Academy of Sciences, 05-110 Jabłonna, Poland Received 7 July 2005; received in revised form 3 October 2005; accepted 4 October 2005 Available online 14 November 2005
Abstract The aim of this study was to determine the developmental changes in the synthesis, storage and release of luteinizing hormone (LH) and folliclestimulating hormone (FSH) in the hypophyseal gonadotroph cells from infancy to peripuberty of ovine ontogeny. An examination has been made in 15 infantile (12-, 15-week-old) and juvenile (22-, 30-week-old) ovary-intact sheep. Histomorphological and functional changes in the adenohypophyseal population of gonadotrophs were assayed with hybridohistochemistry, immunohistochemistry and radioimmunoassay. The percentage of the adenohypophyseal area (PAA) occupied by gonadotrophs containing LHb-mRNA or FSHb-mRNA was highest (P < 0.05) in the 15-week-old sheep compared with the other stages. The gradual increase in the PAA occupied by immunoreactive (ir)-LHb-cells from the 12th to 30th week of age was observed (P < 0.05) and has been interpreted as the increase in the storage of LH. This histomorphological change was accompanied by the gradual increase in the LH pulse frequency from the 15th to 30th week of age (P < 0.05). The PAA occupied by ir-FSHb-cells was extremely high in the infantile sheep, and subsequently, low in the juvenile sheep (P < 0.05). Altogether, similar patterns of pretranslational synthesis of the LHb- and FSHb-subunit but clearly different storage patterns of gonadotrophins were observed. The postnatal development of gonadotrophins seems to be determined by the progressive increase in the storage of LH until peripuberty and by the acute decrease in the storage of FSH during the infantile/juvenile shift. These findings imply the different intrahypophyseal regulation of LH and FSH posttranscriptional processing during the period of transition between infancy and peripuberty in female sheep. # 2005 Elsevier B.V. All rights reserved. Keywords: Sexual development; Gonadotrophic hormones Hybridohistochemistry; Immunohistochemistry; Radioimmunoassay; Female lamb
1. Introduction Sexual development depends on the precise and coordinated functions of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). The adenohypophyseal gonadotroph population, as the site of synthesis, storage and release of LH and FSH, is the morphological basis of the effector mechanism of neuroendocrine functions of the reproductive system (Meeran et al., 2003). The development of the gonadotrophic axis from birth to the peripubertal period has been poorly described in sheep. Very little is known about the postnatal changes of ovine gonadotroph cells’ endocrine functions except the developmental patterns of circulating LH and FSH. These patterns can reflect functional changes of the gonadotrophic axis on the
* Corresponding author. Tel.: +48 22 7824037; fax: +48 22 7742038. E-mail address: [email protected] (M. Wan´kowska). 0891-0618/$ – see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jchemneu.2005.10.002
systemic level and are described by Foster et al. (1975a,b) and Huffman et al. (1987). To our knowledge, the only study related to postnatal changes in the gonadotroph population revealed that in newborn female lambs the percentages of LH and LH– FSH cells do not change, whereas FSH cells percentage increases about two-fold after birth (Messaoud-Toumi et al., 1993). The intrahypophyseal mechanisms underlying the developmental changes in gonadotrophins on the systemic level during the period of transition between infancy and puberty in sheep are unknown. The present work was designed to describe the change from infantile to peripubertal activity of LH and FSH in the adenohypophyseal gonadotroph population of the ovary-intact sheep. The histomorphological developmental changes in the storage and/or synthesis patterns of gonadotrophins in the adenohypophysis were demonstrated by their immunoreactive (ir) or in situ hybridised contents. The immunoreactivity for adenohypophyseal LHb, FSHb and in situ hybridised LHb-
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mRNA or FSHb-mRNA were investigated by immunohistochemical or hybridohistochemical techniques. Additionally, the release of LH into the peripheral circulation was determined by radioimmunoassay. The comparison of adenohypophyseal populations of cells synthesising or storing LH or FSH between postnatal stages of ovine ontogeny may help to describe the nature of the effector mechanism of sexual neuroendocrine development of sheep. 2. Materials and methods 2.1. Animals and tissue preparation The study was performed on purebred Polish Merino female sheep born in the first week of July on the Bieganowo Farm (Poland). Thus, the lambs were born after the summer solstice, i.e. out of season. Lambs were housed in an outdoor environment and left to graze with ewes on pasture for 12 weeks after parturition. Twelve-week-old ovary-intact females were divided randomly into four age groups according to different phases of postnatal ontogenesis— infantile and juvenile periods. Infancy was represented by 12-week-old unweaned sheep (average weight 18.3 1.17 kg; n = 3) and 15-week-old weaned lambs (2 weeks after weaning; average weight 21.1 2.16 kg; n = 4). The juvenile period (adolescence) was represented by 22-week-old prepubertal lambs (2 months around the expected time of puberty; average weight 25.9 2.95 kg; n = 4) and 30-week-old peripubertal lambs (2 weeks around the expected time of puberty; average weight 32.3 2.80 kg; n = 4). The 12-week-old unweaned lambs (sucked their dams one to two times per 24 h) were independent of their mothers in respect of feeding and grazed with flock in the pasture. The slaughter of these lambs was conducted on the local licensed slaughterhouse of the Bieganowo Farm. All the other lambs were transported from the farm to the Institute of Animal Physiology and Nutrition (Jabłonna, Poland) shortly after their weaning at 13 weeks of age. Lambs were maintained indoors in double pens under natural lighting and temperature conditions at 528N and 218E. Animals were fed a diet of commercial concetrates, with hay and water available ad libitum. Every 3 days over a period of 29–30 weeks of age, three consecutive blood samples were taken for determination of progesterone concentrations in the peripheral blood plasma. One day before each experiment day (from 15th to 30th week of age), a jugular venous catheter was inserted and kept with heparinised saline (50 units of heparin/ml in 0.9%, w/v, NaCl). On experiment days, blood samples were taken at 10 min intervals over a 6 h period. Samples were centrifuged and the collected plasma was stored at 20 8C until analysis. During sample collection, the animals were kept in comfortable cages, where they could lie down and have unrestrained access to hay and water. Shortly after the collection, lambs were anesthetised with an i.v. injection of pentobarbitone sodium (20 mg/kg; Biochemie GmbH, Kundl, Austria) and were slaughtered by decapitation in the local licensed slaughterhouse. The ovaries of the 30-week-old lambs, were inspected after slaughter. All procedures were approved by the Local Ethics Committee in Warsaw, according to the Polish Law for the Care and Use of Animals (2 August 1997). Immediately after decapitation, brains were perfused with 1000 ml 0.1 M phosphate buffered saline (PBS; Sigma, St. Louis, USA) and subsequently with 1500 ml 0.1 M PBS containing 4% (w/v) paraformaldehyde (Sigma–Aldrich, Seelze, Germany) and 15% saturated picric acid (MERCK, Darmstadt, Germany) solution (w/v), pH 7.4, via both carotid arteries. The hypophyses were dissected 20 min after the beginning of perfusion and postfixed for 48 h by immersion in the same fixative and washed with 0.01 M PBS. All solutions used for the following steps of tissue preparation and fixation were made up fresh and sterile autoclaved. The hypophyses were dehydrated in graded alcohol, embedded in paraplast (Sigma, St. Louis, USA) and 4 mm sections were cut in the sagittal plane.
2.2. Preparation of probes LHb- and FSHb-sense and antisense riboprobes were produced using previously obtained homologous sheep double-stranded cDNAs (D’Angelo-Bernard
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et al., 1990; Pelletier et al., 1992, 1995). cDNAs (533 bp oLHb-cDNA or 900 bp oFSHb-cDNA) were digoxigenin (DIG) labelled by in vitro transcription with SP6 and T7 RNA polymerase using the DIG-labelling kit from Roche Molecular Biochemicals (Meylan, France) and pGEM-TEasy vectors (Promega, Charbonnieres, France). The specificity of probes was confirmed by the absence of a positive signal in sections hybridised with sense probes (data not shown).
2.3. Hybridohistochemistry The cellular LHb-mRNA and FSHb-mRNA was studied by an in situ hybridisation technique in combination with an IHC detection of DIG. Sections were mounted on slides and coated with 3-aminopropyltriethoxysilane (2% in acetone; Sigma, St. Louis, USA). All sections from every stage were run in a single assay. The non-radioactive in situ hybridisation procedure using DIG-labelled cDNA probes was performed according to Breitschopf et al. (1992) protocol, with minor modifications by Wan´kowska et al. (2002). Briefly, slides were deparaffined, rehydrated, washed in PBS, fixed with 4% paraformaldehyde in PBS and treated with 0.25% acetic anhydride in 100 mM TEA buffer (triethanolamine-Cl; Sigma, St. Louis, USA). Next, the sections were treated with 200 mM HCl, then with 1% proteinase K (ICN, Aurora, OH, USA) dissolved in Tris–EDTA and finally washed in Tris–glycine buffer. Sections were prehybridised at 55 8C in a humid chamber and in situ hybridisation was carried out using DIG-dUTP antisense or sense RNA probes diluted 1:100. After incubation, slides were washed in 2 SSC and treated with 20 mg/ml RNase A (Sigma, St. Louis, USA) in 0.5 M NaCl, 10 mM Tris–HCl (pH 8.0), 10 mM EDTA. Next, the sections were treated with 30% formamide (Sigma, St. Louis, USA) and washed in 2 SSC. The sections were washed in 100 mM Tris–HCl (pH 7.5), 150 mM NaCl, 0.5% blocking reagent (Boehringer, Mannheim, Germany). Next, the sections were incubated with alkaline phosphatase anti-DIG antibody (Boehringer, Mannheim, Germany) diluted 1:500 in the same buffer. Following this, the sections were washed in 100 mM Tris–NaCl (pH 7.5), 150 mM NaCl for 15 min and for 3 min in 100 mM Tris–HCl (pH 9.5), 100 mM NaCl and 50 mM MgCl2. After that the sections were incubated in the last buffer containing 75 mg/ml NBT, 50 mg/ml BCIP (Boehringer, Mannheim, Germany) and 10 mM levamisole (Sigma, St. Louis, USA). The colour reaction was stopped in water and slides were mounted in glycerol-gelatine (Sigma, St. Louis, USA). Controls were prepared using RNase pretreatment. Sections were incubated with RNase (Sigma, St. Louis, USA), 1 unit/100 ml 10 mM Tris–HCl buffer, pH 7.9, per section, just prior to the addition of the hybridisation buffer for 30 min, and then, slides were washed with 4 SSC. Controls did not exhibit any staining (data not shown).
2.4. Immunohistochemistry (IHC) The populations of LH- or FSH-containing cells in the adenohypophysis were determined by means of immunohistochemical light microscopy. All sections taken from all animals were run in a single assay. Hypophyseal sections were deparaffined, rehydrated, washed in 0.01 M PBS, incubated for 30 min in 2% preimmune normal lamb serum in 0.01 M PBS and 30 min in 0.1% hydrogen peroxide (Chempur, Piekary S´la˛skie, Poland) in 0.01 M PBS. Sections were incubated with primary antisera raised in the rabbit: anti-ovine LHb No. I3 diluted 1:1000 and anti-ovine FSHb No. P5 diluted 1:200 for 4 days at 4 8C. Methodological details of their preparation and their specificity were described by Hurbain-Kosmath et al. (1990). After the incubation with primary antibodies, sections were rinsed in 0.01 M PBS, incubated for 2 h at room temperature with the secondary antibody (sheep anti-rabbit Ig [H + L] labelled with peroxidase; Bio-Rad, Steenvoorde, France) diluted 1:40 in 0.1% normal lamb serum in 0.01 M PBS. The colour reaction was developed by incubating sections with 0.05% 30 ,3-diaminobenzidine tetrahydrochloride chromogen (Sigma, St. Louis, USA) and 0.001% hydrogen peroxide in 0.05 M Tris buffer (ICN, Aurora, OH, USA). For control staining, the primary antiserum was replaced with the same dilution of rabbit serum. As an additional control reaction, the inhibition of antihormone serum with its homologous antigen was used. To evaluate the specificity of staining of LH and FSH, corresponding antisera were preadsorbed using synthetic rLH32V02 and rFSH32V02 (National Hormone and Pituitary Program, Torrance, CA, USA). Synthetic antigens and antisera were mixed and
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preincubated for 24 h at 4 8C and then used for the IHC staining instead of the primary antibody. Neither of the controls exhibited any specific staining (data not shown).
by the elaborated small and comparable movements (16 for each section) of screw moving the slide on the microscope stage. The frame size was kept constant for the duration of the image analysis.
2.5. Morphometric analysis
2.6. Determination of LH and progesterone in peripheral blood plasma
A type 104 Nikon projection microscope (Nikon Corporation, Yokohama, Japan) was used for morphometric analyses of hypophyseal sections. The staining was analysed using the ‘‘Lucia’’ image analysis computer system Version 3.51ab (Laboratory Imaging Ltd., Prague, Czech Republic). Analyses were performed under a 40 objective. Pictures of stained sections were projected by a camera (Panasonic KR222, Matsushita Electric Industrial Co., Osaka, Japan) to a colour monitor. Images were adjusted for optimal contrast, fixed at the same brightness level, converted to grey and processed by background subtraction and removal of artifacts. The area fraction parameter defining the percentage of the total area of adenohypophyseal pars distalis that exhibited specific staining was measured. This parameter expresses pool of positively stained cross-sections of gonadotrophs in the total adenohypophyseal cell number. Quantitative analyses were performed for each hypophysis in subareas of pars distalis using a mechanical threshold function to select a range of grey values that were optically identified as positive staining. The analyses of the immunoreaction and/or in situ hybridisation signal in gonadotrophs were made in four sections of each adenohypophysis, using every 40th section (16 fields of 0.0837 mm2 measured in each section). Regions of interest in the pars distalis (for every jointly 64 measured fields) were random—chosen for analysis
The concentration of LH was determined in duplicate 100 ml aliquots by a routine double-antibody radioimmunoassay (RIA) procedure using anti-ovine LH, anti-rabbit-gammaglobuline antisera and bovine LH standard NIH-LH-B6 according to Stupnicki and Madej (1976). The assay detection limit was 0.06 ng/ml sample. The coefficient of variation calculated for control samples at concentration 1 and 5 ng/ml of LH was 10 and 4%, respectively. The concentration of FSH could not be measured due to technical problems. The concentration of progesterone was assayed in duplicate 100 ml aliquots by a direct RIA method routinely used in our laboratory according to procedure described by Stupnicki and Kula (1982) with a sensitivity of 6.2 pg/ml. The intra-assay coefficient of variation was <10%.
2.7. Statistical analyses The quantitative measurements taken from each section for each adenohypophysis were averaged to obtain a mean estimate for each adenohypophysis for each animal. Then, the mean data were pooled to represent the individual age groups. Thus, the data are reported as the mean percentage (for 12-week-old
Fig. 1. Adenohypophyseal gonadotrophs containing in situ hybridised mRNA transcripts encoding LHb (a and b) or FSHb (c and d) subunits in the representative 15week-old lamb (the infantile period; a and c) and 30-week-old lamb (the juvenile period; b and d). Note the higher populations of LHb-mRNA-positive or FSHbmRNA-positive cells in infantile sheep compared with juvenile sheep. Calibration bars 50 mm.
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sheep, n = 3, for other n = 4 per stage) S.E.M. of the total area that exhibited positive and specific immunostaining or hybridohistochemical staining according to developmental stages. Significant effects of postnatal age on the factors studied were assessed by one-way analysis of variance (ANOVA) followed by the post hoc Tukey’s test to determine significant differences between means for individual age groups, using the Statistica 6.0 PL software (StatSoft Inc., Tulsa, OK, USA). Data of RIA are presented as mean (n = 4 per stage) S.D. The mean concentration of LH for individual animals was calculated from the area under the curve (the sum of trapezoid areas between the curve and the abscissa). Pulse characteristic of LH were calculated using the Pulsar Computer Program Version 3.01 developed by Merriam and Wachter (1982) and adapted to operate on an IBM-PC. The cut-off parameters G(n) were settled to a 5% error rate assuming a normal distribution of data. The level of significance for differences between stage groups for data obtained from plasma LH analysis was calculated using the one-way analysis of variance, followed by the post hoc Newman– Keuls test (for mean concentration and amplitude of pulse) or the Kruskal– Wallis test, the non-parametric equivalent of ANOVA for number of pulses. Significance was defined at the P < 0.05 level.
3. Results 3.1. Animals The progesterone concentrations in blood plasma did not exceed 0.5 ng/ml in sheep. Inspection of the 30-week-old sheep ovaries revealed no corpora lutea and at least one dominant ovarian follicle of about 6–7 mm in diameter. 3.2. In situ hybridised LHb-mRNA and FSHb-mRNA No detectable hybridohistochemical signal was observed in sections treated with sense probes for LHb-mRNA or FSHbmRNA (results not shown). Microscopic analysis of the hybridohistochemically stained gonadotrophs showed that within the pituitaries of lambs between 12th and 30th week of age the populations of LHb-mRNA- and FSHb-mRNApositive cells were highest in the 15-week-old sheep compared with the other stages of postnatal development (Fig. 1). Microscopic observations were supported by quantitative computer estimation of the percentages of the adenohypophyseal area occupied by gonadotrophs containing hybridised LHb-mRNA or FSHb-mRNA, which were highest (P < 0.05) in the 15-week-old sheep in comparison with the other investigated stages (Fig. 2). 3.3. Immunoreactive gonadotrophins The population of adenohypophyseal cells immunostained positively for LH increased gradually with progressive developmental stages, up to the expected time of the peripubertal period (Fig. 3). This was reflected by the significant gradual increase in the percentage of adenohypophyseal area occupied by ir-LHb-positive cells within the pituitaries of lambs between the 12th and 30th week of age (from 2.45 0.064 to 5.42 0.140%; P < 0.05; Fig. 4). In contrast to ir-LHb-positive cells, the pool of adenohypophyseal cells immunostained positively for FSH was the highest during the 12th and 15th weeks of age and the lowest during the 22nd and 30th weeks of age (Fig. 3). The percentage of
Fig. 2. The percentage of adenohypophyseal area occupied by gonadotrophs containing in situ hybridised LHb-mRNA (a) or FSHb-mRNA (b) within the pituitaries of lambs between the 12th and 30th week of age. Values are mean S.E.M.; *P < 0.05. Note the similar developmental patterns of the pretranslational synthesis of the both LHb- and FSHb-subunit.
adenohypophyseal area occupied by ir-FSHb-cells during the investigated developmental period was higher (P < 0.05) and relatively stable in the 12- and 15-week-old sheep (3.41 0.088 and 3.45 0.159%; Fig. 4) and subsequently, lower (P < 0.05) and stable in the 22- and 30-week-old sheep (1.26 0.068 and 1.25 0.068%; Fig. 4). 3.4. LH in peripheral blood plasma The LH concentration was higher (P < 0.05) in the 30week-old sheep compared with the earlier stages of postnatal
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Fig. 3. Adenohypophyseal gonadotrophs containing immunoreactive LHb (a and b) or FSHb (c and d) in the representative 12-week-old lamb (the infantile period; a and c) and 30-week-old lamb (the juvenile period; b and d). Note the lower population of LH-containing gonadotrophs and the higher population of FSH-containing gonadotrophs in infantile lamb in comparison with juvenile lamb. Calibration bars 50 mm.
development (Fig. 5a). A gradual increase (P < 0.05) in the LH pulse frequency from the 15th to 30th week of age was observed (from 3 1 to 8 1, number of LH pulses/6 h; P < 0.05; Fig. 5b). No differences were observed in LH pulse amplitude (Fig. 5c). 4. Discussion The subsequent developmental changes in the adenohypophyseal cells producing LH and/or FSH are analysed and compared with hybridohistochemistry, immunohistochemistry and radioimmunoassay. The populations of stained gonadotrophs were higher or lower according to the level of synthesising or releasing cellular activity present until the moment of the tissue’s fixation after decapitation of lambs. 4.1. Developmental patterns of synthesis and storage of LH and FSH The change of lower population of gonadotrophs stained by hybridohistochemistry into higher one has been interpreted as the increase in the gonadotrophic b subunits
transcriptional synthesis and/or stability of mRNAs. The regulation of gonadotrophin subunits’ gene expression and translation may be influenced by alterations in stability and half-life of mRNA (see for review, Crawford and McNeilly, 2002). The pretranslational synthesis of b subunits of LH and FSH in the gonadotroph population was highest during the shift from the infantile to the juvenile period compared with the other stages of postnatal development. Similar developmental changes of gonadotrophin b subunit mRNAs were assessed in the female rat (Zapatero-Caballero et al., 2004). Namely, LHb-mRNA and FSHb-mRNA levels as determined by Northern blot analysis rose slowly during the infantile period and thereafter, the levels of both mRNAs fell during the juvenile period until peripuberty (Zapatero-Caballero et al., 2004). Thus, similar developmental patterns of pretranslational synthesis of the LHb- and FSHb-subunit were observed during the postnatal development of female sheep. These pretranslational patterns were accompanied by differential posttranslational regulation of LH and FSH storage. The change of lower population of adenohypophyseal gonadotrophs stained by IHC into higher one has been
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Fig. 4. The percentage of adenohypophyseal area occupied by gonadotrophs containing immunoreactive LHb (a) or FSHb (b) subunit within pituitaries of lambs between the 12th and 30th week of age. Values are mean S.E.M.; * P < 0.05. Note the clearly different storage patterns of gonadotrophins according to investigated stages of ontogeny.
interpreted as the increase in the storage, whereas the opposite change has been interpreted as the decrease in the accumulation of hormone. The storage of LH increased gradually until the expected time of the peripubertal period, whereas the storage of FSH was extremely high during infancy and extremely low during the juvenile period. Thus, clearly different storage patterns of LH and FSH were observed during the period of transition between infancy and peripuberty in the female sheep. Previously, dynamic postnatal modifications of the heterogeneity of LH and FSH storage were observed in the
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hypophysis of the female rat (Jansen, 1982). The numbers of gonadotrophs per pituitary tissue and the amounts of LH and FSH in pituitary cells increased during the infantile period. Then, the number of LH cells remained high until puberty, whereas the number of FSH cells decreased (Jansen, 1982). Furthermore, in the male rhesus monkey, the pituitary gonadotroph population increased, and a large number of monohormonal FSH gonadotrophs were likely to become bihormonal during the juvenile–adult transition period (Meeran et al., 2003). Meeran et al. (2003) suggested that these findings attest that gonadotrophic cells can be multipotent, switching their hormonal identity in systemic response to different environmental influences. Altogether, similar patterns of the LHb- and FSHb-subunit pretranslational synthesis, but clearly different storage patterns of gonadotrophins were observed in the postnatally developing female sheep. The results obtained in the current investigation on sheep support the discussed above observations related to the rat and monkey (Jansen, 1982; Meeran et al., 2003; ZapateroCaballero et al., 2004). This similarity led us to define and employ of the new one division in the postnatal sexual development of sheep, on the basis of patterns of the both gonadotrophins’ synthesis and the specific storage of FSH. As in the rat and monkey, the ovine postnatal sexual development is divided now into: (i) the infantile period of increase in the both gonadotrophins’ synthesis and the high storage of FSH (after the neonatal period, to the transitional stage beyond the time of weaning jointly) and (ii) the juvenile period of decrease in the both gonadotrophins’ synthesis and low storage of FSH (beginning no earlier than after the time of weaning and represented by prepuberty and peripuberty). The terminology and duration of the different stages of postnatal sexual development of sheep have been defined firstly by Foster et al. (1975a) on the basis of patterns of circulating LH. The postnatal secretion of LH in the female sheep was often pulsatile and by 11 weeks of age serum LH attained levels far above adult baseline. Further, during sexual maturation and early adulthood concentrations of circulating LH and its baseline levels remained stable and greater than those in the mature cycling ewe (Foster et al., 1975a,b). Thus, the tonic LH secretion during the course of postnatal ontogeny of female sheep has been divided by Foster et al. (1975a) into the following three phases: (i) a neonatal period (the first 2–5 weeks) of low secretion; (ii) a period of high secretion during sexual maturation and early adulthood; (iii) a period of low secretion during true adulthood. However, these patterns can reflect functional changes of the gonadotrophic axis only on the systemic level. Thus, in the current study, the new one division in the postnatal sexual development of female sheep has been defined on the basis of intrapituitary changes in the synthesis and storage of both gonadotrophins. The observations obtained in this investigation led to the hypothesis that regulatory mechanisms responsible for divergent LH and FSH secretion during the postnatal development of female sheep may be explained by the differential posttranscriptional regulation of both gonadotrophins.
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Fig. 5. The plasma LH concentration (a), frequency (b) and amplitude (c) in ovary-intact female lambs during transition from the infantile to the peripubertal period of ontogeny. Values are mean (for every n = 4) S.D.; *P < 0.05. Note the higher plasma LH concentration in the 30-week-old sheep, the gradual increase in the LH pulse frequency from the 15th to 30th week of age and no differences in the LH pulse amplitude according to investigated stages of ontogeny.
4.2. Regulatory mechanisms of divergent posttranscriptional processing of LH and FSH In the current study, the plasma LH concentration was stable during the shift from infancy to the juvenile period and during prepuberty. Then, it was enhanced at the expected time of peripuberty. Furthermore, the gradual increase in LH pulse frequency accompanied by stable pulse amplitude during the transition period between infancy and the expected time of the peripubertal period was observed. It is generally accepted that an increase in the frequency of episodic LH secretion is a key event leading to the onset of ovarian cycles in the lamb, whereas an increase in pulse amplitude must occur just before the LH surge (Huffman et al., 1987). The lambs from the present investigation were born out of season, i.e. after the summer solstice, what differs from most of the literature published on the topic. However, the current results of pulse frequency and concentration of LH in the ovary-intact Merino lambs, which are a less photoperiodic breed, can be compared to findings obtained from ovariectomised highly seasonal Suffolk lambs, born near the spring equinox and maintained in a permissive sequence of photoperiods in the presence of estradiol replacement (Ebling et al., 1990). The current findings describing regulatory mechanisms of divergent posttranscriptional processing of LH and FSH cannot be compared to results related to sheep born in spring, due to the fact that there are not any results related to the synthesis or storage of both gonadotrophins obtained from postnatally developing female lambs born near the spring equinox. It should be pointed out that the current study simply proposes intrahypophyseal mechanisms, which might account for the postnatal development of the ovine gonadotrophic axis. In this study, during the juvenile period, the decrease in the adenohypophyseal population of cells containing LHb-mRNA accompanied by the increase in the LH-containing cell
population and in the plasma LH pulse frequency were observed. Thus, we hypothesize that changes in the histological feature of gonadotroph population, dependent on the increase in the number of LH-containing cells, can represent the intrahypophyseal posttranscriptional mechanisms that differentially regulate the secretion of both gonadotrophins during the transition period between infancy and adulthood in the female sheep. Furthermore, the postnatal development of gonadotrophins seems to be determined by the acute and characteristic decrease in the storage of FSH during the ovine infantile/juvenile transitional stage. This is in agreement with a suggestion of Meeran et al. (2003), that, the secretion of both LH and FSH throughout sexual development could be differentially regulated by changes in the proportion of gonadotroph subtypes within the total pituitary gonadotroph population. Moreover, Crawford and McNeilly (2002) suggested that in the female sheep, the majority of synthesised LH seems to be laid aside into storage (McNeilly et al., 1991) and to be released upon extracellular stimulation, directly mimicking the pulsatile secretion of gonadotrophin-releasing hormone (GnRH) (Clarke and Cummins, 1982). Thus, a characteristic of the GnRH/LH secretory pathway is the ability of LH protein to be stored (Crawford and McNeilly, 2002). Crawford and McNeilly (2002) revealed that the secretory pattern of LH does not reflect its transcriptional rate, but is related to posttranslational intrapituitary mechanisms due to the storage and transportation of secretory granules within the gonadotrophs of the ewe (Thomas and Clarke, 1997). This intracellular mechanism has been suggested by Currie and McNeilly (1995) to switch non-releasing gonadotrophs into a potentially releasable state. Some LH is additionally released constitutively (McNeilly et al., 1991), possibly to maintain basal levels of LH (Crawford et al., 2000). In view of discussed above discrepancies, the findings of the current study imply the existence of the histomorphological feature of the intrahypo-
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physeal regulation of posttranscriptional processing and release of LH during the transition from infantile to the peripubertal activity of gonadotroph population in the female sheep. The data obtained in the present investigation reveal the increase in the population of cells containing FSHb-mRNA accompanied by the extremely high number of FSH-containing cells during the infantile period. During the juvenile period, the decrease in the population of cells containing FSHb-mRNA accompanied by the extremely low number of FSH-containing cells was observed. Concentrations of FSH in the blood plasma were not determined due to technical problems, but it is perhaps pertinent to notice that Foster et al. (1975b) made a note of increase in the plasma FSH between the 15th and 20th week of postnatal age. Thereafter, the decrease in the plasma FSH between the 20th and 30th week of postnatal age was observed in ovary-intact female lambs (Foster et al., 1975b). Crawford and McNeilly (2002) suggested that in contrast to the secretion of LH, the release of FSH is primarily constitutive (Farnworth et al., 1988, 1995), i.e. FSH is trafficked to the site of exocytosis shortly after synthesis and is released. Thus, there exists a strong relationship between the level of pituitary FSHb-mRNA and plasma FSH, which is representative for constitutively secreted hormones (Crawford and McNeilly, 2002). In conclusion, the findings of current study imply the existence of the relationship between the pretranslational synthesis and storage of FSH in contrast to the relationship between the storage and pulse frequency of LH during the postnatal sexual development of female sheep. Altogether, the data obtained in the present study and these discussed above, led to the conclusion that it is more probable that the differential regulation of both gonadotrophins in the postnatally developing female sheep is on the level of posttranscriptional processing and release rather than on the level of gene expression. The suggested requirement for sexual maturation is the change in the histomorphological feature of gonadotroph population, dependent on the increase in the number of LH-containing cells. 4.3. Conclusions Similar patterns of pretranslational synthesis of the LHband FSHb-subunits but clearly different storage patterns of both gonadotrophins were observed in the postnatally developing female sheep. These observations lead to the suggestion that the regulatory mechanism responsible for divergent LH and FSH secretion during the postnatal development of female sheep could be explained by the more probable different regulation on the level of storage and release rather, than on the level of gene expression. It is suggested that the histomorphological changes dependent upon: (i) the gradual increase in the storage of LH according to progressive developmental stages and (ii) the acute decrease in the storage of FSH during the infantile/juvenile shift, could represent the intrahypophyseal posttranscriptional mechanisms that differentially regulates the both gonadotrophins’ secretion during the period of transition between infancy and puberty in the female sheep. Altogether, mechanisms of postnatal sexual development in the female sheep, that are
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dependent upon the coordinated function of LH and FSH, are likely to operate within the adenohypophysis and could have at least partly the histomorphological feature. The findings of this study are novel for ruminants and support observations in the rat and monkey. Acknowledgments The authors are grateful to the Mrs. Ewa Skrzeczyn´ska and Mrs. Anna Misztal for technical assistance. They also express their thanks to Dr. Raymond Counis for kind provision with LHb- and FSHb-sense and antisense probes. This study was supported by the Polish Committee for Scientific Research, Grant No. 6 PO6D 008 20. References Breitschopf, H., Suchanek, G., Gould, R.M., Colman, D.R., Lassman, H., 1992. In situ hybridization with digoxigenin-labelled probes: sensitive and reliable detection method applied to myelinating rat brain. Acta Neuropathol. 84, 581–587. Clarke, I.J., Cummins, J.T., 1982. The temporal relationship between gonadotropin releasing hormone (GnRH) and luteinizing hormone (LH) secretion in ovariectomized ewes. Endocrinology 111, 1737–1739. Crawford, J.L., McNeilly, A.S., 2002. Co-localisation of gonadotrophins and granins in gonadotrophs at different stages of the oestrous cycle in sheep. J. Endocrinol. 174, 179–194. Crawford, J.L., Currie, R.J.W., McNeilly, A.S., 2000. Replenishment of LH stores of gonadotrophs in relation to expression, synthesis and secretion of LH after the preovulatory phase of the sheep oestrus cycle. J. Endocrinol. 167, 453–463. Currie, R.J.W., McNeilly, A.S., 1995. Mobilization of LH secretory granules in gonadotrophs in relation to gene expression, synthesis and secretion of LH during the preovulatory phase of the sheep oestrous cycle. J. Endocrinol. 147, 259–270. D’Angelo-Bernard, G., Moumni, M., Jutisz, M., Counis, R., 1990. Cloning and sequence analysis of the cDNA for the precursor of the beta subunit of ovine luteinizing hormone. Nucleic Acid Res. 18, 2175. Ebling, F.J.P., Kushler, R.H., Foster, D.L., 1990. Pulsatile LH secretion during sexual maturation in the female sheep: photoperiodic regulation in the presence and absence of ovarian steroid feedback as determined in the same individual. Neuroendocrinology 52, 229–237. Farnworth, P.G., Robertson, D.M., de Kretser, D.M., Burger, H.G., 1988. Effects of 31 kilodalton bovine inhibin on follicle-stimulating hormone and luteinizing hormone in rat pituitary cells in vitro: actions under basal conditions. Endocrinology 122, 207–213. Farnworth, P.G., Thean, E., Robertson, D.M., 1995. Ovine anterior pituitary production of follistatin in vitro. Endocrinology 136, 4397–4406. Foster, D.L., Jaffe, R.B., Niswender, G.D., 1975a. Sequential patterns of circulating LH and FSH in female sheep during the early postnatal period: effect of gonadectomy. Endocrinology 96, 15–21. Foster, D.L., Lemons, J.A., Jaffe, R.B., Niswender, G.D., 1975b. Sequential patterns of circulating luteinizing hormone and follicle-stimulating hormone in female sheep from early postnatal life through the first estrous cycles. Endocrinology 97, 985–994. Huffman, L.J., Inskeep, E.K., Goodman, R.L., 1987. Changes in episodic luteinizing hormone secretion leading to puberty in the lamb. Biol. Reprod. 37, 755–761. Hurbain-Kosmath, I., Berault, A., Noel, N., Polkowska, J., Bohin, A., Jutisz, M., Leiter, E.H., Beamer, W.G., Bedigian, H.G., Davisson, M.T., Harrison, D.E., 1990. Gonadotropes in a novel rat pituitary tumor cell line, RC-4B/C. Establishment and partial characterisation of the cell line. In Vitro Cell. Dev. Biol. 26, 431–440. Jansen, H.G., 1982. Distribution and number of gonadotrophic cells in the pituitary gland of prepubertal female rats. J. Endocrinol. 94, 381–388.
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