Macroheterogeneity in follicle-stimulating hormone (FSH) β-subunit N-glycosylation results in distinct FSH glycoforms. Hypoglycosylated FSH21 is the abundant and more bioactive form in pituitaries of females under 35 years of age, whereas fully glycosylated FSH24 is less bioactive and increases with age. To investigate whether the shift in FSH glycoform abundance contributes to the age-dependent decline in oocyte quality, the direct effects of FSH glycoforms on folliculogenesis and oocyte quality were determined using an encapsulated in vitro mouse follicle growth system. Long-term culture (10-12 days) with FSH21 (10 ng/ml) enhanced follicle growth, estradiol secretion and oocyte quality compared with FSH24 (10 ng/ml) treatment. FSH21 enhanced establishment of transzonal projections, gap junctions and cell-to-cell communication within 24 h in culture. Transient inhibition of FSH21-mediated bidirectional communication abrogated the positive effects of FSH21 on follicle growth, estradiol secretion and oocyte quality. Our data indicate that FSH21 promotes folliculogenesis and oocyte quality in vitro by increasing cell-to-cell communication early in folliculogenesis, and that the shift in in vivo abundance from FSH21 to FSH24 with reproductive aging may contribute to the age-dependent decline in oocyte quality.

Female reproductive aging is characterized by a rise in follicle-stimulating hormone (FSH) levels during peri-menopause. N-linked glycans are co-translationally attached to the Asn⁷ and Asn²⁴ residues on the FSHβ subunit. Differences in the number of N-glycans on the FSHβ subunit result in distinct glycoforms: hypo-glycosylated (FSH^21/18, glycans absent on either Asn²⁴ or Asn⁷, respectively) or fully-glycosylated (FSH²⁴, glycans present on both Asn⁷ and Asn²⁴). The relative abundance of FSH glycoforms changes with advanced reproductive age, shifting from predominantly FSH^21/18 in younger women to FSH²⁴ in older women. Previous in vitro studies in granulosa cell lines and in vivo studies using Fshb-null mice showed these glycoforms elicit differential bioactivities. However, the direct effects of FSH glycoforms on the mouse ovarian follicle have not yet been determined. In this study, we isolated secondary follicles from pre-pubertal mice and treated them with 20- or 100 ng/mL purified recombinant FSH glycoforms for 1 h or 18–20 h. Analysis of phosphorylated PKA substrates showed that glycoforms were bioactive in follicles following 1-h treatment, although differential bioactivity was only observed with the 100 ng/mL dose. Treatment of follicles with 100 ng/mL of each glycoform also induced distinct expression patterns of FSH-responsive genes as assessed by qPCR, consistent with differential function. Our results, therefore, indicate that FSH glycoforms are bioactive in isolated murine follicles. Abstract Female reproductive aging is characterized by a rise in follicle-stimulating hormone (FSH) levels during peri-menopause. N-linked glycans are co-translationally attached to the Asn⁷ and Asn²⁴ residues on the FSHβ subunit. Differences in the number of N-glycans on the FSHβ subunit result in distinct glycoforms: hypo-glycosylated (FSH^21/18, glycans absent on either Asn²⁴ or Asn⁷, respectively) or fully-glycosylated (FSH²⁴, glycans present on both Asn⁷ and Asn²⁴). The relative abundance of FSH glycoforms changes with advanced reproductive age, shifting from predominantly FSH^21/18 in younger women to FSH²⁴ in older women. Previous in vitro studies in granulosa cell lines and in vivo studies using Fshb-null mice showed these glycoforms elicit differential bioactivities. However, the direct effects of FSH glycoforms on the mouse ovarian follicle have not yet been determined. In this study, we isolated secondary follicles from pre-pubertal mice and treated them with 20- or 100 ng/mL purified recombinant FSH glycoforms for 1 h or 18–20 h. Analysis of phosphorylated PKA substrates showed that glycoforms were bioactive in follicles following 1-h treatment, although differential bioactivity was only observed with the 100 ng/mL dose. Treatment of follicles with 100 ng/mL of each glycoform also induced distinct expression patterns of FSH-responsive genes as assessed by qPCR, consistent with differential function. Our results, therefore, indicate that FSH glycoforms are bioactive in isolated murine follicles.

In the mouse, two distinct populations of Leydig cells arise during testis development. Fetal Leydig cells arise from a stem cell population and produce T required for masculinization. It is debated whether they persist in the adult testis. A second adult Leydig stem cell population gives rise to progenitor-immature-mature adult type Leydig cells that produce T in response to LH to maintain spermatogenesis. In testis of adult null male mice lacking either only LH (Lhb^−/−) or LHR (Lhr^−/−), mature Leydig cells are absent but fetal Leydig cells persist. Thus, it is not clear whether other ligands signal via LHRs in Lhb null mice or LH signals via other receptors in the absence of LHR in Lhr null mice. Moreover, it is not clear whether truncated LHR isoforms generated from the same Lhr gene promoter encode functionally relevant LH receptors. To determine the in vivo roles of LH-LHR signaling pathway in the Leydig cell lineage, we generated double null mutant mice lacking both LH Ligand and all forms of LHR. Phenotypic analysis indicated testis morpho-histological characteristics are identical among double null and single mutants which all showed poorly developed interstitium with a reduction in Leydig cell number and absence of late stage spermatids. Gene expression analyses confirmed that the majority of the T biosynthesis pathway enzyme-encoding mRNAs expressed in Leydig cells were all suppressed. Expression of thrombospondin-2, a fetal Leydig cell marker gene was upregulated in single and double null mutants indicating that fetal Leydig cells originate and develop independent of LH-LHR signaling pathway in vivo. Serum and intratesticular T levels were similarly suppressed in single and double mutants. Consequently, expression of AR-regulated genes in Sertoli and germ cells were similarly affected in single and double mutants without any evidence of any additive effect in the combined absence of both LH and LHR. Our studies unequivocally provide genetic evidence that in the mouse testis, fetal Leydig cells do not require LH-LHR signaling pathway and a one-to-one LH ligand-LHR signaling pathway exists in vivo to regulate adult Leydig cell lineage and spermatogenesis.