Influence of ovarian hormones on endocrine activity of gonadotroph cells in the adenohypophysis of lambs during the postnatal transition to prepuberty

Animal Reproduction Science 121 (2010) 84–93

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Influence of ovarian hormones on endocrine activity of gonadotroph cells in the adenohypophysis of lambs during the postnatal transition to prepuberty ∗ ´ , Jolanta Polkowska, Tomasz Misztal, Katarzyna Romanowicz Marta Wankowska Department of Endocrinology, The Kielanowski Institute of Animal Physiology and Nutrition, Polish Academy of Sciences, Instytucka 3, 05-110 Jabłonna, Poland

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Article history: Received 11 March 2010 Received in revised form 6 May 2010 Accepted 12 May 2010 Available online 20 May 2010 Keywords: Luteinizing hormone Follicle-stimulating hormone Adenohypophysis Postnatal development Steroid feedback Female sheep

a b s t r a c t There is juvenile hiatus during maturation of larger mammals with relatively long life spans. Using histomorphological and functional criteria we describe the feedback mechanisms which could play a role in the regulation of the gonadotrophic axis during the postnatal transition to the quiescent prepubertal period in sheep. The aim of this study was to determine the influence of ovarian factors on the endocrine activity of gonadotroph cells, the site of synthesis, storage and release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH), in adenohypophyses of weanling and weaned prepubertal lambs. The examination was made in (i) 9-week-old infantiles, suckling lambs undergoing weaning, ovary-intact (OVI) and ovariectomised (OVX) at the 6th week of age, and (ii) 16-week-old juveniles OVI and OVX at the 12th week of age (n = 5 per group). Changes in gonadotrophs were assayed with hybridohistochemistry, immunohistochemistry and radioimmunoassay. The percentage of the adenohypophyseal area (PA) occupied by gonadotrophs containing LH␤-mRNA and immunoreactive for LH␤ was lower (P < 0.05), whereas the PA occupied by cells containing FSH␤-mRNA and immunoreactive for FSH␤ was higher (P < 0.05) in the 16-week-old OVI lambs in comparison with the 9-week-old ones. The mean concentration and basal level of LH in the peripheral blood plasma were greater (P < 0.05) in the 16-week-old OVI lambs in comparison with the 9-week-old group, whereas the circulating FSH was not different. In the OVX 9-week-old lambs, the PA occupied by gonadotrophs containing LH␤-mRNA and the plasma LH concentration, basal level, pulse frequency and amplitude were greater (P < 0.05), whereas the PA occupied by cells immunoreactive for LH␤ was lower (P < 0.05) in comparison with the OVI group. In the OVX 16-week-old lambs, the PA occupied by gonadotrophs containing LH␤-mRNA and immunoreactive for LH␤, the LH plasma concentration, basal level and pulse frequency were (P < 0.05) greater in comparison with the OVI group. The PA occupied by gonadotrophs containing FSH␤-mRNA and the plasma FSH concentration were greater (P < 0.05) in the OVX 9- and 16-week-old lambs in comparison with the OVI ones. The ovariectomy had no effect on the PA occupied by cells immunoreactive for FSH␤ in both age stages. In conclusion, ovarian factors may play inhibitory role in regulating both LH and FSH synthesis rate and release and stimulatory role in regulating LH storage in adenohypophyseal gonadotrophs in infantile lambs. In lambs at the beginning of the juvenile period, ovarian factors may play only inhibitory role in regulating both LH and FSH synthesis and release and LH storage. The effects of ovarian hormones on the gonadotrophin storage, i.e. releasable pools in adenohypophyseal cells, are specific for LH,

∗ Corresponding author. Tel.: +48 22 7653 315; fax: +48 22 7653 302. ´ E-mail addresses: [email protected] (M. Wankowska), [email protected] (J. Polkowska), [email protected] (T. Misztal), [email protected] (K. Romanowicz). 0378-4320/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2010.05.006

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no such effects are apparent on FSH in lambs during the postnatal transition to prepuberty. Thus, the initiation of the juvenile period in female sheep is a function of change of the stimulatory role of ovarian hormones in regulating LH storage to the inhibitory one. © 2010 Elsevier B.V. All rights reserved.

1. Introduction The hypophyseal gonadotrophic cells, as the site of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion, are the morphological setting for the endocrine mechanisms that prepare an organism for reproduction. The postinfantile transition to adolescence in mammals involves the neuroendocrine states which evoke ´ significant changes in secretion of hormones (Wankowska ´ et al., 2006, 2008b; Wankowska and Polkowska, 2006, 2009). These endocrine changes lead to the full presentation of juvenile phenotype, i.e. physiological readiness to sexual maturation (for review see Veldhuis et al., 2006). The most important feature of juvenile phenotype is an increase in the secretion of LH under changes in the post-translational processing and neuroanatomy of gonadoliberin (GnRH) in the preoptic area-hypothalamus ´ from the infantile to the pubertal pattern (Wankowska et ´ al., 2008a; Wankowska and Polkowska, 2009). The development of the gonadotrophic axis from birth to the juvenile period in female sheep, i.e. during the first 15 weeks of postnatal life, has been poorly described. Information concerning endocrine functions of gonadotroph cells are limited to the patterns of circulating LH and FSH (Foster et al., 1975a,b). These patterns can reflect functional changes of the gonadotrophic axis on the systemic level. Throughout the first 5 weeks of postnatal life, the levels of serum LH are diminished. With the onset of pulsatile LH secretion, beginning 11 weeks after birth, circulating LH increases to levels greater than those observed in adults (Foster et al., 1975b). After the neonatal increase in the concentration of FSH to levels characteristic for adult ewe, the next increase in the levels of serum FSH is observed from 15 to 20 weeks of age (Foster et al., 1975b). In this regard, in newborn female lambs the percentages of LH and LH–FSH cells do not change, whereas FSH cells percentage increases about twofold after birth (MessaoudToumi et al., 1993). Fourteen weeks later, i.e. during the initiation of the juvenile period, the gonadotrophic mechanisms involve the different intrahypophyseal regulation of LH and FSH post-transcriptional processing. The development of secretory processes in gonadotrophs during the juvenile period seems to be determined by the decrease in the storage of FSH and the increase in the storage of ´ LH until peripuberty (Wankowska and Polkowska, 2006). The hypophyseal endocrine mechanisms underlying the changes in gonadotrophins on the systemic level throughout the infantile period, i.e. the transition period between the neonatal and juvenile period, in sheep are yet to be identified and described. In “precocious species” (e.g. primates), the periods of prenatal and neonatal activation of gonadotrophic axis may be equivalent to the postnatal periods of activation

in “altricial species” (e.g. laboratory strains of rodents) i.e. mammals with a short life span and rapid growth and development. In this regard, there is juvenile hiatus during neuroendocrine maturation of larger mammals with relatively long life spans (for review see Ebling, 2005). The reactivation of reproductive development after the quiescent prepubertal period is determined by the reduction in sensitivity of the hypothalamo-hypophyseal unit to inhibitory feedback of the gonadal steroid hormones (Claypool and Foster, 1990; Veldhuis et al., 2006). Thus, it is essential to understand which aspect of the regulation of LH and FSH reflects the feedback action of gonadal hormones before juvenile readiness to sexual maturation in such precocious species as the sheep, inputs from the central nervous system and/or some other aspect? In the present study we focus on the possibility that the patterns of LH and FSH secretion after the neonatal period in female lambs might be related to the influence of the ovarian hormones on changes in releasable pools of gonadotrophin in adenohypophyseal cells. 2. Materials and methods 2.1. Animals, management and experimental design All procedures were approved by the Local Ethics Committee affiliated to Warsaw Agriculture University (number of opinion 29/2006), according to the Polish Law for the Care and Use of Animals (2 August 1997), and thus were conducted in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki) for animal experiments. The 3- to 4-year-old ewes of the highly seasonal Polish Longwool breed (features of the breed in Rasali et al., 2006) were mated naturally at a commercial sheep facil˛ ity (Samokleski Farm, Poland) and were transported to the Institute of Animal Physiology and Nutrition (Jabłonna, Poland) in September. Sixteen female lambs from twin pregnancy and four from singleton pregnancy were born in February. The animals were fed a diet which provided 100% of the National Research Institute of Animal Production recommendations for pregnancy and lactation in ewes and suckling period in lambs (Norms, 1993). The lambs were penned with dams indoors, in individual pens, under not controlled lighting and temperature conditions present at 52◦ N latitude and 21◦ E longitude. Ten lambs were weaned at 65th day of age and were fed ad libitum hay and twice daily a complete pelleted concentrate ration supplemented with vitamins and minerals and containing 17% of protein (norms for fattening period in lambs). This diet assured the optimal tempo of growth. Four-week-old lambs were divided randomly into four groups (n = 5, one from singleton pregnancy and four from

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twin pregnancy per group) according to different phases of postnatal ontogeny, infancy and the beginning of the juvenile period. Infancy was represented by 9-week-old weanlings (63rd day of age) ovary-intact (OVI) and ovariectomised (OVX) at 39th day of age. They suckled their mothers 3–4 times per 24 h. The juvenile period was represented by 16-week-old lambs (112th day of age) OVI and OVX at 88th day of age. These lambs should begin pubertal ovarian cycles approximately 14 weeks later. The ovariectomy was performed under ketamine/pentobarbitone anaesthesia (8–10 and 10–20 mg/kg body weight, respectively; intravenous injection). The weaned OVI and OVX lambs were maintained in two separate large pens. A day before collection of blood plasma a jugular venous catheter was inserted and kept with heparinized saline. Blood samples were collected every 10 min for a 5 h (from 08:30 to 13:20 h) in 5- (37th day of age), 9-, 12- (86th day of age) and 16-week-old OVI lambs and 9- and 16-week old OVX lambs. During collection, the lambs were kept in their pens where they could lie down and had unrestrained access to hay, water and/or dams. Blood samples were centrifuged in heparinized tubes and the plasma was stored at −20 ◦ C until LH and FSH assays. At the day of dissection, 9-week-old and 16-week-old OVI and OVX lambs were anesthetized with an intravenous injection of pentobarbitone sodium (20 mg/kg; Biochemie GmbH, Kundl, Austria) and decapitated (from 13:30 to 14:30 h). 2.2. Tissue preparation The tissue samples for histological assessment were prepared using the clinical standard of formaldehyde fixation with minor modifications and paraffin embedding. Immediately after decapitation, the brains were perfused via the carotid arteries with 0.1% sodium nitrite in 1500 ml 0.1 M phosphate buffered saline (PBS) (Sigma, St. Louis, USA) and then with 1500 ml 0.1 M PBS containing 4% (w/v) paraformaldehyde (Sigma–Aldrich, Seelze, Germany), pH 7.4. Hypophyses were removed from the cranium, postfixed at room temperature for 24 h by immersion in this same fixative, dehydrated in graded alcohol, embedded in paraplast (Sigma, St. Louis, USA) and sliced into 5 ␮m sections in the sagittal plane. 2.3. Preparation of probes and hybridohistochemistry Cellular LH␤ and FSH␤ mRNA was studied by in situ hybridisation in combination with immunohistochemical detection of digoxigenin (DIG). Riboprobes were produced using previously obtained homologous sheep doublestranded cDNAs (D’Angelo-Bernard et al., 1990; Pelletier et al., 1992, 1995). The 533 bp oLH␤-cDNA or 900 bp oFSH␤cDNA were 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). The non-radioactive in situ hybridisation procedure using DIG-labeled cDNA probes was

´ performed according to the Wankowska et al. (2002). Briefly, slides were 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, Ohio, USA) dissolved in Tris-EDTA and finally washed in Tris-glycine buffer. Sections were subsequently pre-hybridized and in situ hybridisation with DIG-dUTP anti-sense or sense probes diluted 1:100 was carried out at 55 ◦ C in a humid chamber. 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 and in 100 mM Tris–HCl (pH 9.5), 100 mM NaCl and 50 mM MgCl2 . Finally, the sections were incubated in 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. Control sections were incubated with RNase, 1 unit/100 ␮l 10 mM Tris–HCl buffer, pH 7.9, for 30 min, just prior to the addition of the hybridisation buffer. Controls did not exhibit any staining (data not shown). 2.4. Immunohistochemistry The LH- and FSH-containing cells in the adenohypophysis were identified by the peroxidase labelled antibody method. Sections were 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 ´ askie, ˛ Sl Poland) in 0.01 M PBS. The sections were incubated with primary rabbit anti-sheep LH␤ No. I3 diluted 1:1000 and anti-sheep FSH␤ No. P5 diluted 1:200 for 4 days at 4 ◦ C. The antibodies were prepared in C.N.R.S. (Gif sur Yvette, France). Methodological details of their preparation and specificity are described by Hurbain-Kosmath et al. (1990). After incubation with primary antibodies, sections were incubated for 2 h at room temperature with the 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% 3 3-diaminobenzidine tetrahydrochloride chromogen (Sigma, St. Louis, USA) and 0.001% hydrogen peroxide in 0.05 M Tris buffer (ICN, Aurora, Ohio, USA). For control staining, the primary antiserum was replaced with the same dilution of rabbit serum and anti-hormone serum was inhibited with its homologous antigen. To evaluate the specificity of LH and FSH staining, corresponding antisera were preabsorbed using synthetic rLH32V02 and rFSH32V02 (National Hormone and Pituitary Program, Torrance, CA, USA). Synthetic antigens and

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antisera were mixed and preincubated for 24 h at 4 ◦ C and then used for the immunohistochemical staining instead of the primary antibodies. Neither of the controls exhibited any specific staining (data not shown). 2.5. Image analyses A type ECLIPSE 80i Nikon projection microscope, Nikon DS-Fi1 camera and NIS-Elements Basic Research image analysis computer system version 2.32 (Nikon Corporation, Tokyo, Japan) were used for histological analyses of the adenohypophysis under objective lens 40×. The crosssectional area fraction parameter was measured to define the percentage of the total area of the hypophyseal pars distalis (PA) that exhibited specific staining. This parameter describes the dimension of the population of cells positively stained for LH␤ mRNA, FSH␤ mRNA, LH␤ or FSH␤. The analyses of the histochemical reaction in gonadotrophs were performed using a mechanical threshold function to select a range of values that were optically identified positive for staining. They were made in four sections of each adenohypophysis, using every 40th section (16 fields of 0.08215 mm2 measured in each section). Methodological details of the morphometric analyses had been described more precisely in our previous published stud´ ´ ies (Wankowska et al., 2006; Wankowska and Polkowska, 2006). 2.6. Determination of LH and FSH in peripheral blood plasma The concentration of LH was determined in duplicate 100-␮l aliquots by a routine double-antibody radioimmunoassay (RIA) procedure using anti-ovine LH, anti-rabbit-gammaglobulin antisera, and bovine LH standard NIH-LH-B6 (Stupnicki and Madej, 1976). The assay detection limit was 0.312 ng/ml of sample. The intra- and interassay coefficients of variations calculated for control samples at concentrations of 5 and 1 ng/ml of LH were 4 and 10%, respectively. Plasma FSH concentration was estimated by doubleantibody RIA, using anti-ovine-FSH (teri. anti-oFSH) and anti-rabbit-gammaglobulin antisera. The anti-FSH, as well as the FSH standard (teri. oFSH and teri. FSH ig) was kindly supplied by Dr. L.E. Reichert Jr. (Tucker Endocrine Research Institute LLC, Atlanta, Georgia, USA). The assay sensitivity was 1.56 ng/ml and the intra- and inter-assay coefficients of variation were 3.3 ± 1.5 and 11.3 ± 2.2%, respectively. 2.7. Statistical analyses The measurements of area fraction taken for each factor from each section for each adenohypophysis were averaged to obtain a mean estimate for the hypophysis of each animal. Then, the above-mentioned mean data were pooled to represent the individual groups of animals. Thus, the data are reported as the mean of percentage of the total area that exhibited staining according to studied groups (n = 5 per group). The effects of age and ovariectomy on the factors studied were assessed by one-way analysis of variance (ANOVA) followed by the post hoc

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Tukey’s test and the Student’s t-test to determine significant differences between means for individual groups, using the Statistica 6.0 PL software (StatSoft Inc., Tulsa, OK, USA). The mean concentration of LH or FSH for individual animals was calculated from the area under the curve (the sum of trapezoid areas between the curve and the abscissa). The effects of the age and ovariectomy on plasma hormone concentrations were analysed within groups (between infancy and the beginning of the juvenile period) and between groups (during infancy and the beginning of the juvenile period) by ANOVA followed by the post hoc Tukey’s test. Pulse characteristics of LH were calculated using the Pulsar Computer Program developed by Merriam and Wachter (1982) and adapted to operate on an IBM-PC. The cut-off parameters G(n) were set as 5% error rate assuming a normal distribution of data. Analysis was performed individually for every lamb and included the entire sampling period. The significance of differences in LH pulse frequency (defined as the number of identified pulses per collecting period) within groups was assayed by the Wilcoxon test and between groups by the Mann–Whitney test. The levels of significance for differences between data of amplitude and peak length of LH pulses within and between groups were calculated by the nonparametric ANOVA rank Kruskal–Wallis test. All data are expressed as means ± SEM. Significance was defined the P < 0.05.

3. Results 3.1. Histochemical detection of LHˇ mRNA, FSHˇ mRNA, LH and FSH The optically detected population of LH␤ mRNApositive cells was greater in 9-week-old than in 16-weekold OVI lambs (Fig. 1), whereas the population of FSH␤ mRNA-positive cells was optically greater in 16-weekold than in 9-week-old OVI lambs (Fig. 1). Populations of LH␤ mRNA- and FSH␤ mRNA-positive cells were optically greater in OVX lambs than in OVI ones from both age stages (Fig. 1). These qualitative results were reflected by the higher (P < 0.05) PA occupied by gonadotrophs containing in situ hybridised mRNAs (Fig. 2A and B). The optically detected population of gonadotrophs immunoreactive for LH and the PA occupied by these cells were greater in 9-week-old than in 16-week-old OVI lambs and in 9-week-old OVI lambs compared to OVX ones (P < 0.05; Figs. 1 and 2C). At the 16-week-old stage, an inverse situation was observed in OVI lambs versus OVX lambs (Fig. 2C). In the case of cells immunoreactive for FSH, their population was optically greater in 16-week-old than in 9-week-old OVI lambs (Fig. 1), what was reflected by the higher (P < 0.05) PA occupied by gonadotrophs containing FSH (Fig. 2D). The population of FSH␤-immunoreactive gonadotrophs was similar in adenohypophyses of OVI and OVX lambs from the 9-week-old (Fig. 1) and 16-week-old stage, what was reflected by the similar PA occupied by gonadotrophs containing FSH (Fig. 2D).

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Fig. 1. Gonadotroph cells containing in situ hybridised mRNA transcripts encoding LH␤- and FSH␤-subunit or immunoreactive for LH␤ and FSH␤ subunits in the adenohypophyseal pars distalis of the representative ovary-intact (OVI) versus ovariectomised (OVX) infantile lamb and juvenile OVI lamb. PA, the percentage of area occupied by cross-sections of stained cells. Note a great population of gonadotrophs containing LH␤ mRNA and FHS␤ mRNA and a small population of LH-containing gonadotrophs in the OVX infantile lamb compared with the OVI one. In the juvenile lamb note a great population of FSH-containing gonadotrophs.

3.2. LH and FSH in peripheral blood plasma The plasma LH concentration and basal level was lower in 5- and 9-week-old OVI lambs in comparison with 12and 16-week-old ones (P < 0.05; Fig. 3). The frequency of LH pulses increased from 5 until 12 weeks of age (P < 0.01) and decreased at 16 weeks of age (P < 0.05). The LH pulse amplitude was lower in 5-week-old OVI lambs in comparison with the later age stages (P < 0.05) and the peak length was lowest in 5-week-old OVI lambs and highest in 16-weekold ones (P < 0.01; Fig. 3). The plasma FSH concentration was higher in 16-week-old OVI lambs in comparison with the previous age stages (P < 0.05; Fig. 4). In OVX 9- and 16-week-old lambs the LH plasma concentration, basal level and frequency of LH pulses (P < 0.05; Fig. 3) and plasma concentration of FSH (P < 0.01; Fig. 4) were greater in comparison with the OVI groups. The LH pulse amplitude was higher only in OVX 9-week-old lambs in comparison with the OVI group (P < 0.05; Fig. 3). The

peak length was lower in OVX 16-week-old lambs than in OVI ones (P < 0.05; Fig. 3). In OVX lambs the mean concentration of FSH was huge in comparison with the mean concentration of LH in both age stages (84.9 ± 10.06 versus 14.0 ± 0.80 ng/ml in 9-week-old group and 42.5 ± 3.46 versus 11.7 ± 0.92 ng/ml in 16-week-old group; P < 0.001; Figs. 3 and 4). The plasma concentration of both LH and FSH was higher in 9-week-old OVX lambs than in 16-week-old ones (P < 0.05; Figs. 3 and 4). 4. Discussion The study elucidates the mechanisms of ovarian hormones feedback at a cellular level in adenohypophysis during the transition from the neonatal to juvenile period in sheep. The converse links demonstrated between presence of ovaries and dimension of population of LH-containing gonadotrophs in studied stages provide evidence that ovarian factors may play a role in regulating LH storage during

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Fig. 2. The percentage of adenohypophyseal area (cross-sectional area fraction) occupied by the gonadotrophs containing in situ hybridised LH␤mRNA (A) or FSH␤-mRNA (B) and immunoreactive for the LH␤ (C) or FSH␤ (D) subunit within the hypophyseal pars distalis of infantile (9-week-old) and juvenile (16-week-old) lambs ovary-intact (OVI) and ovariectomised (OVX) 4 weeks before the hypophyses dissection. Values are means (for every n = 5) ±SEM; *P < 0.05, **P < 0.01, ***P < 0.001; a and b: a statistical significant difference for OVI and OVX versus OVI lambs, respectively.

infancy and at the beginning of the juvenile period. The low accumulation of LH in absence of ovaries in infantiles suggests the stimulatory role, whereas the high storage of LH after ovariectomy in juvenile lambs suggests the inhibitory role. In contrast to LH, the ovariectomy had no effect on the accumulation of FSH in gonadotrophs in both age stages during the postnatal transition to prepuberty. However, the increase in the dimension of subpopulation of FSH-containing gonadotrophs was accompanied by the decrease in the dimension of subpopulation of LHcontaining cells at the beginning of the juvenile period. As in the case of lambs undergoing the transition to the juvenile period after the period of weaning, in newborn female lambs the percentage of FSH-containing gonadotrophs increases after birth, whereas LH-containing cells percentage do not change (Messaoud-Toumi et al., 1993). These findings suggest that in precocious species the secretion of both LH and FSH from birth to prepuberty should be differentially regulated by neonatal and postweaning changes in the proportion of gonadotroph subtypes within the total adenohypophyseal gonadotroph population, as was found previously for prepubertal and pubertal lambs ´ ´ (Wankowska and Polkowska, 2006; Wankowska et al., 2008a) and rhesus monkey (Meeran et al., 2003). In con-

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trast to precocious species, in altricial species such as the rat, the numbers of gonadotrophs per hypophyseal tissue and the amounts of both LH and FSH in hypophyseal cells increase during infancy until the juvenile period. Then the number of LH cells remains high until puberty (Jansen, 1982). Altogether, the heterogeneity in the dimensions of gonadotroph subpopulations containing LH and FSH in the postnatal transition to prepuberty is related to some adjustment of the ovarian hormones feedback loop, i.e. the change of the positive feedback effect on the number of gonadotrophs containing LH to the negative one. Such homeostatic feedback mechanism seems to be specific for the regulation of the gonadotrophic axis during the beginning of the quiescent prepubertal period in the female sheep, which is a representative of larger mammals with longer life span. These effects of the age and ovariectomy on the gonadotrophs containing LH and FSH were accompanied by the different effects on the accumulation of mRNA for ␤ subunits of LH and FSH. A feature of the transition from the infantile to juvenile period is the decrease in the dimension of gonadotroph population containing LH␤ mRNA and increase in the case of cells containing FSH␤ mRNA. However, the LH␤ and FSH␤ mRNA abundance in absence of ovaries in both infancy and at the beginning of the juvenile period suggests the inhibitory role of ovarian hormones in regulating the transcription rate of both LH␤ and FSH␤. The lamb and rat differ in their LH synthesis rate during postnatal development until the juvenile period. In female rats, LH␤ and FSH␤ mRNA contents rise throughout infancy to the juvenile period (Zapatero-Caballero et al., 2004). However it should be noted that in both the weanling lamb and infantile rat FSH␤ mRNA is dramatically regulated in the adenohypophysis. In rats the source of this regulation does not appear to be GnRH (Wilson and Handa, 1997) but probably gonadal steroid hormones (Wilson and Handa, 1998). The previous studies on adult ewes (Baratta et al., 2001; Clarke, 2002; DiGregorio and Nett, 1995; for review see Nett et al., 2002) and infantile female rats (Wilson and Handa, 1998) indicate that gonadotrophs are capable of responding directly to gonadal hormones, and thus may play a role in the selective regulation of LH and FSH expression and secretion in vivo. It was previously suggested, that the negative feedback of estradiol on LH and FSH secretion mainly targets the bihormonal gonadotroph cells and occurs, at least in part, directly at the hypophysis level in adult ewes (Molter-Gerard et al., 2000). Altogether, the discordant changes in the transcription rate are similar to the changes in accumulation of LH and FSH in the ovine adenohypophyseal endocrine cells during the postnatal transition to prepuberty. The decrease in the accumulation of LH␤ mRNA and increase in the accumulation of FSH␤ mRNA after the infantile period are under negative feedback effects of gonadal hormones in female lambs. The patterns of circulating hormones image the described changes in the gonadotrophin transcription rate. Circulating LH was uniformly low in infantile stages and then uniformly high beyond the period of weaning. It was previously suggested that low tonic LH secretion, presumably in the form of slow pulses, is necessary for development or maintenance of ovarian function before puberty

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Fig. 3. (A) The peripheral blood plasma luteinizing hormone (LH) mean concentration, basal level, pulse frequency, pulse amplitude, peak length in weanling (5- and 9-week-old) and weaned prepubertal (12- and 16-week-old) lambs ovary-intact (OVI) and ovariectomised (OVX) 4 weeks before blood collection. Values are means (for every n = 5) ±SEM; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; a–c: a statistical significant difference for OVI, OVX versus OVI and OVX versus OVX lambs, respectively. (B) The pulsatile pattern of LH release in individual lambs.

in lambs (Foster et al., 1986). The FSH plasma levels were uniform until the 11th week of age and there was prepubertal increase in the circulating FSH in 16-week-old lambs. The stages of the developmental increase in serum LH and FSH levels correspond with the stage of the juvenile period beginning after the weaning period in our experimental model. However, the release of both gonadotrophins during the infantile–juvenile transition period seems to be

under negative feedback effects of gonadal hormones. The changes in the patterns of both gonadotrophins release are similar in lambs and infantile rats, as is in the case of the synthesis rate. The plasma LH and FSH rose to higher levels during infantile development until the beginning of the juvenile period and estradiol likely is the major candidate in stimulation of FSH secretion in the infantile female rat (Herath et al., 2001). In this regard, estradiol is capable of

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Fig. 4. (A) The mean concentration of peripheral blood plasma follicle-stimulating hormone (FSH) in weanling (5- and 9-week-old) and weaned prepubertal (12- and 16-week-old) lambs ovary-intact (OVI) and ovariectomised (OVX) 4 weeks before blood collection. Values are means (for every n = 5) ± SEM; *P < 0.05, **P < 0.001, ***P < 0.0001; a–c: a statistical significant difference for OVI, OVX versus OVI and OVX versus OVX lambs, respectively. (B) The pattern of FSH release in individual lambs.

altering secretion of FSH and LH in the absence of GnRH in adenohypophyseal cells from anestrous ewes (Baratta et al., 2001). However, an explanation of the dramatic increase in FSH levels after ovariectomy, as compare with more moderate increase in LH in the current study, could lie in the paracrine or endocrine influences of factors that may selectively affect the secretion of only FSH. It is conceivable that ovariectomy suppressed inhibin, the protein produced by developing ovary follicles and affecting FSH

in gonadotroph cells (Burger et al., 2001; Farnworth, 1995; Gregg et al., 1991). It should be noted, that in the female rat the inhibin regulation of hypophyseal FSH secretion, through its negative feedback, begins to operate during the transition from the infantile to the juvenile prepubertal period (Herath et al., 2001). Altogether, the endocrine transition from the neonatal to juvenile period in female sheep is related to similar regulation on the level of LH and FSH synthesis and release and different regulation on the level

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of their storage. The increase in the LH and FSH synthesis and release rate after the neonatal period is under negative feedback effects of gonadal hormones in female lambs. In lambs during the postnatal transition to prepuberty, the rate of FSH release is related to the rate of synthesis, i.e. it is constitutive. The release of FSH in mammals is primarily constitutive (Farnworth et al., 1988, 1995). It is trafficked to the site of exocytosis shortly after synthesis, and released (Crawford and McNeilly, 2002). The direct relationship between the level of hypophyseal FSH␤ mRNA and plasma FSH is representative for constitutively secreted hormones (Brooks et al., 1992; Crawford and McNeilly, 2002; McNeilly, 1988). In contrast to FSH, the secretory pattern of LH does not reflect its transcriptional rate but rather the translational processing in female lambs which begin the juvenile period. This postinfantile secretory pattern does not reflect LH post-translational intrahypophyseal mechanisms due to the storage of secretory granules within the gonadotrophs, as is in the case ´ of older prepubertal and pubertal lambs (Wankowska and ´ Polkowska, 2006; Wankowska et al., 2008a) and in ewes during the oestrous cycle (Crawford et al., 2000; Crawford and McNeilly, 2002). The cellular mechanism observed in weanlings in contrast to juvenile lambs is similar to the positive feedback action of oestrogen which mobilizes LHcontaining, but not FSH-containing secretory granules in gonadotrophs of cycling ewes (Currie and McNeilly, 1995; Thomas and Clarke, 1997). It should be remained, that for the operation of a regulatory/storage pathway for the secretion of LH is required GnRH; no such mechanism appears for FSH (McNeilly et al., 1991). Altogether, whilst a pivotal feature of the GnRH/LH secretory pathway in the ovine quiescent prepubertal period is ability of LH pro´ tein to be stored (Wankowska and Polkowska, 2006, 2009; ´ Wankowska et al., 2008a), an important feature of the GnRH/LH axis during postnatal transition to prepuberty is the ability of LH to be synthesized and released. 5. Conclusions The increase in both the LH and FSH synthesis rate and release is actively inhibited, whereas increase in the storage of FSH in adenohypophyseal cells is passive under ovarian feedback effects during the transition from the infantile to juvenile period in lambs. An endocrine feature of gonadotroph cells during initiation of the juvenile period is decrease in accumulation of LH. From a cellular perspective of gonadal–hypophyseal feedback loop, the beginning of the juvenile period is a function of change of the stimulatory role of ovarian hormones in regulating LH storage, i.e. releasable pools of LH in adenohypophyseal cells, to the inhibitory one. Such homeostatic feedback mechanisms seem play an important role in the regulation of the gonadotrophic axis during the postnatal transition to the quiescent prepubertal period in sheep. Funding This study was supported by Grant No. N311 004 32/0224 (Ministry of Science and Higher Education, Poland).

Acknowledgments ´ The authors are grateful to Mrs. Ewa Skrzeczynska and Mrs. Anna Misztal for technical assistance and would like to express their thanks to vet Józef Rutkowski for surgery. They also express their thanks to Dr. Raymond Counis for kind provision with LH␤- and FSH␤-cDNAs.

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