Hormones and Reagents. Rat and ovine INSL3 were chemically synthesized and characterized as described (1). Rat INSL3 was used for functional testing, whereas ovine INSL3 was appended with a biotin residue on the N terminus of the A chain for ligand binding studies. GnRH antagonist ganirelix acetate, human LH, and human FSH were obtained from Organon (West Orange, NJ), and human chorionic gonadotropin (hCG) was from Ayerst Laboratories (Philadelphia). ¹²⁵I-streptavidin and streptavidin conjugated to horseradish peroxidase were purchased from Amersham Biosciences, cilostamide was from Calbiochem, and pregnant mare serum gonadotropin (PMSG), forskolin, Pertussis toxin, collagenase, and 1-methyl-3-isobutylxanthine (MIX) were from Sigma. Sprague–Dawley rats were obtained from Simonsen Laboratories (Gilroy, CA). Animal care was consistent with institutional and National Institutes of Health guidelines.

RNA Analyses. Ovaries were obtained from immature rats at different ages or after treatment with gonadotropins, whereas testes were obtained during development and after treatment with a GnRH antagonist with or without hCG or INSL3. Total RNA from rat gonads was extracted by using the RNeasy purification kit (Qiagen, Valencia, CA) before Northern blotting. Rat orthologs for LGR8 and INSL3 were identified in GenBank (accession nos. AC098990 and NM053680, respectively) to design primers for RT-PCR to yield an LGR8 probe of 230 bp and an INSL3 probe of 422 bp for Northern blotting and in situ analyses. For in situ hybridization studies, tissues were fixed at 4°C for 6 h in 4% paraformaldehyde in PBS, followed by immersion in 0.5 M sucrose in PBS overnight. Cryostat sections (7 m m thick) were mounted on microscope slides coated with poly(L)-lysine (Sigma Chemical Co.) and stored at –80°C until analyzed. The hybridization procedure has been described (2). In brief, sections were pretreated serially with 0.2 M HCl, 2× SSC, pronase E (0.125 mg/ml), 4% paraformaldehyde, acetic anhydride in triethanolamine, and dehydrated in ascending grades of ethanol. The antisense and sense probes were labeled with [³⁵S]UTP [1,000 Ci/mmol (1 Ci = 37 GBq); Amersham Pharmacia] and sections were hybridized overnight at 45°C in 50% formamide, 0.3 M NaCl, 10 mM Tris•HCl, 5 mM EDTA, 1× Denhardt’s solution, 10% dextran sulfate, 1 m g/ml carrier transfer RNA, and 10 mM DTT. Following ribonuclease A (20 m g/ml) treatment at 37°C for 30 min, posthybridization washing was performed to a final stringency of 0.1× SSC. Slides were dipped into NTB-2 emulsion (Kodak) and exposed at 4°C for 1 week before development. The slides were stained with hematoxylin/eosin and mounted with DPX Mountant (Electron Microscopy Sciences, Ft. Washington, PA).

GV stage oocytes, cumulus cells, and mural granulosa cells were collected from ovaries of PMSG-treated immature rats at 48 h after treatment. Cumulus-enclosed oocytes (CEOs) were obtained from 26-day-old female rats injected with 15 units of PMSG. At 48 h after hormonal treatment, ovaries were dissected in Leivovitz’s L-15 medium (Invitrogen) before puncturing the largest follicles to obtain CEOs. Following treatment with hyaluronidase for 1-2 min, denuded oocytes were separated from cumulus cells. Mural granulosa cells were obtained separately by puncturing preovulatory follicles, followed by the removal of CEOs. RT-PCR and nested PCR for GV stage oocytes, cumulus cells, and mural granulosa cells were described (3). Because of the low number of GV stage oocytes under study, hemi-nested PCR was needed to achieve optimal amplification. The primers for rat LGR8 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were as follows: LGR8, sense 5'-CTGGATCGTGGTTTTTTTCCTTCCGGT-3' and antisense 5'-CATTATACTCGTCCCCAAGGGCTATTT-3'; hemi-nested sense primer, 5'-CAGTTTAAGTGAAGAGTCATCTGTCC-3'; and for GAPDH, sense 5'-TGAAGGTCGGTGTCAACGGATTTGGC-3' and antisense 5'-CATGTAGGCCATGAGGTCCACCAC-3'. After 30 cycles of PCR reactions, the hemi-nested primer was paired with the antisense primer for another 30 cycles. Quantification of INSL3 transcripts was performed by using SmartCycler (Cepheid, Sunnyvale, CA) according to the manufacturer’s protocol. The primers and hybridization probes for real-time PCR of INSL3, LH receptor, FSH receptor, and b -actin were as follows: INSL3, sense 5'-CTTCCTCACCAGGCTTCTCA-3' and antisense 5'-AATAATTGACACACACCACCTGA-3'; probe 5'-6-carboxy-fluorescein (FAM)-TGTGCACCGCTGCTGTCTCACT-6-carboxy-tetramethyl-rhodamine (TAMRA); LH receptor, sense 5'-ATGGCCATCCTCATCTTCACA-3', antisense 5'-TGGCACAAGAATTGACAGGA, probe, 5'-FAM-TTGCCATCTCAGCTGCCTTCAA-TAMRA-3'; FSH receptor, sense 5'- GTGGTCATCTGTGGCTGCTA-3', antisense 5'-TCTTGGTGCGCTTGATGAG-3', probe 5'FAM-CCACATCTACCTCACAGTGAGGAATCC-TAMRA-3'; b -actin, sense 5'-TCTGTGTGGATTGGTGGCTCTA-3', antisense 5'-CTGCTTGCTGATCCACATCTG-3', probe 5'-FAM-CTTGCCCACAGCCTTGGCAGC-TAMRA-3'.

To determine the copy number of the target transcripts, different cDNAs were used to generate calibration curves by plotting the threshold cycle (Ct) vs. the known copy number for each plasmid template. The copy numbers for target samples were determined according to the calibration curve. To correct for differences in RNA quality and quantity between samples, data were normalized by dividing the copy number of the target cDNA by that of b -actin.

Evaluation of Oocyte Maturation. For evaluating oocyte maturation, CEOs were obtained from PMSG-treated rats and cultured as described (4). CEOs were collected in the L15 medium containing 0.25 mM of MIX within 20 min of ovary retrieval, washed twice in L15 medium without MIX, and transferred to the test medium without MIX. The CEOs were cultured with or without rat INSL3 or other hormones. For controls, the CEOs were cultured in L15 medium containing MIX. At the end of culture, the occurrence of germinal vesicle breakdown (GVBD) in the oocytes was examined after removing cumulus cells surrounding the CEOs by using a small-bore pipette under Hoffman modulation contrast microscopy (Nikon, Tokyo). For preovulatory follicle cultures, follicles were excised from the ovary after PMSG treatment for 48 h. The follicles, 20–30 per vial, were cultured with or without different doses of rat INSL3 or LH (5 m g/ml) in the L15 medium. Some follicles also were treated with Pertussis toxin (1 m g/ml) to suppress Gi activity. The vials were flushed at the start of the culture with O₂/N₂ (1/1) and cultured at 37ºC with gentle shaking. After culture, individual CEOs were dissected from each follicle to examine the occurrence of GVBD. For cAMP determination, 30–40 denuded oocytes obtained from preovulatory follicles were cultured with or without rat INSL3 (10 nM) in L15 medium containing 10 m M of cilostamide, a specific inhibitor of phosphodiesterase 3. After treatment, oocytes were collected in 2 m l of medium and freeze/thawed to break the cells before acetylation of samples with triethylamine and acetic anhydride (3:1). Intraoocyte cAMP levels were determined by RIA as described (5).

To study the effect of INSL3 on oocyte maturation in vivo, intrabursal injection was performed (4). Immature rats were primed with PMSG (15 IU) and, at 48 h later, lightly anesthetized, and one of the ovaries was exteriorized through a small lumbosacral incision. Rat INSL3 (2 m g per 0.1 ml of PBS) or PBS were injected through a 30-gauge needle threaded into the ovarian bursa via the adjoining fat pad. After injection, the ovary was returned to the abdominal cavity and skin was clipped. For positive control, hCG (10 units) was administrated by i.p. injection. At different intervals after treatment, CEOs were collected by puncturing the largest ovarian follicles to examine the occurrence of oocyte GVBD.

Preparation of Seminiferous Tubules and Interstitial Cells. Seminiferous tubular cells and interstitial cells were isolated by a published method (6). Briefly, testes were decapsulated and incubated for 15 min in PBS containing 0.25% collagenase with occasional shaking. After collecting interstitial cells released from the preparation, seminiferous tubules were washed and incubated with 0.25% trypsin to disperse tubular cells. The resulting cell suspension was filtered through a 100-m m nylon mesh to remove cell aggregates and tissue debris after which the tubular cells were collected by centrifugation. Purity of interstitial and tubular cells was assessed based on expression of transcripts for the LH receptor expressed in Leydig cells and the FSH receptor expressed in Sertoli cells (LH receptor/b -actin relative levels: 867 ± 95 × 10^–4 for interstitial cells, 2.3 ± 0.1 × 10^–4 for tubular cells; FSH receptor/b -actin relative levels: 26.7 ± 4.9 × 10^–5 for tubular cells, 1.3 ± 0.2 × 10^–5 for interstitial cells). For testing INSL3 actions, tubular cells were treated with different hormones or forskolin for 12 h before cAMP determination. Some cells were also pretreated for 6 h with Pertussis toxin to suppress Gi activity. Total cAMP in each well was measured in triplicate by specific RIA. For INSL3 binding studies, cells were suspended in D-PBS containing 1% BSA and incubated with biotinylated ovine INSL3 (10 nM) with or without increasing doses of rat INSL3 at 4ºC for 24 h. After incubation, cells were centrifuged and washed twice with 1% BSA/PBS before incubation with ¹²⁵I-streptavidin (400,000 cpm per tube) for 1 h at 4ºC. After three washings, radioactivity in the pellets was determined (7). For testing the binding of INSL3 to LGR8, recombinant LGR8 was expressed. Human 293T cells derived from embryonic kidney fibroblasts were maintained in DMEM/Ham’s F-12 (DMEM/F12) supplemented with 10% FBS, 100 m g/ml penicillin, 100 m g/ml streptomycin, and 2 mM L-glutamine. These cells (2 × 10⁶ per culture) were transfected as described (7) and used for binding studies.

Apoptosis Analyses. For in vivo studies on INSL3 action on the testis, male Sprague–Dawley rats at 28 days of age were treated s.c. with the GnRH antagonist ganirelix acetate (250 m g/kg/day) to suppress pituitary gonadotropin secretion. Some animals were treated with the GnRH antagonist together with s.c. injections of hCG (75 units/day) or rat INSL3 (1 m g/12 h). Animals were killed 5 days later, and organ weight was determined. Testes were stored at –70ºC before RNA or DNA extraction, or fixed in Bouin’s solution for in situ labeling of DNA ends. DNA from whole testis was isolated and quantitated with spectrophotometry at 260 nm. Aliquots of DNA (500 ng) from each sample were labeled at 3' ends with ³²P-dideoxy-ATP (3,000 Ci/mmol; Amersham Biosciences) by using terminal transferase (25 units per sample; Boehringer-Mannheim) (8). Labeled samples were fractionated through 2% agarose gels. After electrophoresis, gels were dried for 2 h in a slab-gel dryer without heat and exposed to Kodak X-Omat AR film at –70° C. DNA fragmentation was visualized by autoradiography and quantitated by b -counting of low molecular weight (<15 kb) DNA fragments. For in situ DNA 3'-end labeling, fixed testicular tissues were embedded in paraffin and cut into 3-m m sections. To detect apoptotic DNA fragmentation, the Fluorescein in situ cell death detection kit (Roche Diagnostics) was used according to manufacturer’s instructions, and sections were counterstained with propidium iodide (0.01 mg/ml, Sigma).

Statistical Analysis. Statistical analysis was carried out by using the Mann–Whitney U test for paired comparison or the one-way ANOVA coupled with Bonferroni t test for multiple group comparison. Results are presented as mean ± SEM of at least three separate experiments.

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