Oocyte quality is enhanced by hypoglycosylated FSH through increased cell-to-cell interaction during mouse follicle development

Follicle-stimulating hormone (FSH) is a gonadotropin produced by the anterior pituitary that is essential for female reproduction (Kumar et al., 1997). FSH secretion is under the control of gonadotropin-releasing hormone (GnRH) and the activin-inhibin-follistatin signaling network, resulting in positive and negative paracrine/endocrine regulation within the reproductive axis. FSH binds and signals via FSH receptors (FSHRs), which are exclusively expressed by ovarian granulosa cells (GCs) of primary, secondary and antral ovarian follicles (Bao and Garverick, 1998; Nimrod et al., 1976; Oktay et al., 1997; O'Shaughnessy et al., 1996). FSH-responsive genes in GCs are estimated at >3000 and include well-known targets such as luteinizing hormone receptor (Lhcgr), Inhibin α (Inhba) and inhibin β (Inhbb), aromatase (Cyp19a1), and cyclin D2 (Ccnd2) (Herndon et al., 2016). Fshb and Fshr knockout female mice are infertile and exhibit arrested folliculogenesis at the pre-antral stage, indicating that folliculogenesis occurs in both FSH-independent and -dependent phases (Abel et al., 2000; Dierich et al., 1998; Kumar et al., 1997). Follicle growth can proceed to the secondary stage in the absence of FSH. However, the action of FSH during early stages of folliculogenesis likely enhances follicle and oocyte parameters, as demonstrated by positive effects of FSH stimulation on pre-antral follicle growth and survival in in vitro systems as well as the negative effect of hypophysectomy on pre-antral follicle numbers in vivo (Adriaens et al., 2004; Kreeger et al., 2005; McGee et al., 1997).

FSH is a heterodimeric glycoprotein consisting of α- and β-subunits, of which the α-subunit is shared with other pituitary and placental glycoprotein hormones, including luteinizing hormone (LH), thyroid stimulating hormone (TSH) and human chorionic gonadotropin (hCG). Thus, the β-subunit (FSHβ) confers the specific biological activity of FSH. Human FSH exhibits microheterogeneity of the oligosaccharide structure in both α- and β-subunits and macroheterogeneity of FSHβ N-glycosylation (reviewed by Bousfield and Harvey, 2019). FSH glycosylation is physiologically regulated, with micro- and macroheterogeneity exhibiting modulation during the pubertal transition, ovarian cyclicity, and with age in females (Bousfield et al., 2014a; Chappel et al., 1983; Creus et al., 1996; Phillips et al., 1997; Wide, 1982; Wide and Bakos, 1993; Zambrano et al., 1995). FSH microheterogeneity, as characterized by chromatofocusing of isoelectric fractions, results in FSH isoform populations with distinct receptor binding affinities and biological activities (Cerpa-poljak et al., 1993; Miller et al., 1983). However, mass spectrometry has demonstrated extensive diversity of glycan microheterogeneity in recombinant, pituitary and urinary preparations (Bousfield et al., 2014a, 2015; Butnev et al., 2015). Furthermore, individual glycans do not separate into distinct isoelectric fractions (Bousfield et al., 2008). Thus, the correlation between glycan microheterogeneity and the differential activity of isoelectric FSH isoform populations remains poorly understood. In contrast, macroheterogeneity of N-glycosylation of FSHβ results in distinct glycoforms with well-characterized differential bioactivities (Bousfield et al., 2018).

N-glycosylation on either one or both FSHβ asparagine residues (Asn⁷ and Asn²⁴) results in various glycoforms, including fully glycosylated FSH²⁴ and hypoglycosylated FSH²¹ and FSH¹⁸. However, only FSH²⁴ and FSH²¹ are readily detectable in human pituitary preparations (Bousfield et al., 2007). Fully and hypoglycosylated FSHβ glycoforms have differential FSHR binding kinetics and biological activities in vitro and in vivo (Agwuegbo et al., 2021; Bousfield et al., 2014b; Butnev et al., 2015; Hua et al., 2021; Jiang et al., 2015; Wang et al., 2016; Zariñán et al., 2020). Recombinant hypoglycosylated FSH, consisting of FSH²¹ and FSH¹⁸ (FSH^21/18), exhibits higher activity in competitive binding assays, twofold higher FSHR saturation, and faster FSHR association kinetics compared with FSH²⁴ (Bousfield et al., 2014b; Butnev et al., 2015). This translates to an increase in in vitro bioactivity of hypoglycosylated FSH, as FSH^21/18 enhances the magnitude and rate of FSHR signaling cascade activation in the KGN GC tumor cell line (Jiang et al., 2015). Furthermore, hypoglycosylated FSH shows greater efficiency in promoting follicle growth in vivo compared with fully glycosylated FSH (Hua et al., 2021). When injected into postnatal day 5 Fshb null male mice, hypoglycosylated FSH increases testes tubule size and germ cell number, and upregulates FSH-responsive genes more effectively than fully glycosylated FSH in vivo (Wang et al., 2016).

FSH glycoform abundance is modulated with age (Bousfield et al., 2018). Analysis of FSH glycoform composition of female pituitaries across the aging spectrum demonstrates that FSH²¹ exhibits higher abundance in reproductively young females under 35 years of age, whereas FSH²⁴ predominates after menopause (Bousfield et al., 2007, 2014a, 2018). This shift in glycoform abundance from the highly bioactive FSH²¹ to the lower activity of FSH²⁴ occurs coordinately with the age-dependent decline in ovarian function associated with decreased oocyte quantity and quality (Duncan et al., 2018). Although age-associated defects in the developmental competence of the fully grown oocyte have been well characterized (Cimadomo et al., 2018; Pan et al., 2008), how age-dependent factors influence the active growth phase of follicular development remains elusive. Such knowledge is important as this stage of follicular development is a crucial determinant of gamete quality (Duncan and Gerton, 2018). FSH glycoforms have differential activities on cultured pre-antral ovarian follicles with respect to modulation of FSH-responsive genes (Simon et al., 2019), growth, survival, and apoptotic gene expression (Johnson et al., 2022). However, the mechanism underlying the differential effects of FSH glycoforms on in vitro cultured follicles and the subsequent impact on the resulting oocytes remain unknown.

We hypothesized that FSH glycoforms have differential abilities to promote folliculogenesis, which may result in differences in oocyte quality. To address this, we utilized an alginate-based, encapsulated in vitro mouse follicle growth system (eIVFG) (Fig. 1A) that supports complete follicle development from the early secondary to the antral follicle stage. Fully grown follicles resulting from this system can be induced to ovulate and produce mature metaphase II (MII)-arrested eggs (Converse et al., 2023). The direct effects of various concentrations (1-100 ng/ml) of purified FSH²¹ and FSH²⁴ were assessed for their effect on follicle growth and survival through 12 days of culture. Furthermore, follicles treated with 10 ng/ml FSH glycoform concentration were induced to ovulate ex vivo, enabling assessment of gamete quality. Finally, transcriptomic and cell-interaction analyses in the early stages of follicle growth identified the cellular mechanism by which age-specific FSH glycoforms modulate differential folliculogenesis and oocyte quality parameters.

To confirm that Fshr is expressed in the early secondary follicles utilized in this study (95-125 µm), we performed quantitative real-time PCR (RT-qPCR) using primers spanning exons 1-3 and exons 9-10 of Fshr (Fig. 1B,C). In qPCR with both proximal and distal exon-specific primers, we found that Fshr was expressed in isolated early secondary (non-treated) follicles, and this expression was enriched compared with whole ovaries from prepubertal wild-type mice. The enrichment of Fshr expression in follicles relative to whole ovaries is expected given the heterogeneity of cell types in the ovary that are not FSH responsive. Fshr expression was absent in ovaries of Fshr-null mice and pituitaries of wild-type mice, as expected. Overall, this indicated that granulosa cells of early secondary follicles express Fshr at this point in follicle development and would be FSH responsive and therefore suitable for use in our study design. Importantly, we were very stringent in selecting follicles within a narrow size range to avoid any confounding differences in responsiveness as a result of follicle size.

To determine the effects of FSH glycoforms on follicle survival and growth, we separately cultured early secondary follicles in media containing either glycoform at two different concentrations (1 and 10 ng/ml). Follicle survival was >70% for all FSH glycoform treatments on day 10 (D10) of culture (Fig. 2A,B). In contrast, only 20% of follicles cultured without FSH (control) survived, which demonstrates that the transition to FSH dependence occurs in the culture system (Fig. 2A,B). By D12 of culture, all glycoform treatments sustained survival above 70% except for 1 ng/ml FSH²⁴ and the FSH negative control treatments, in which survival was only 43±34.45% and 0%, respectively (Fig. 2C). In follicles that survived, all treatments resulted in follicle growth over the duration of culture, including FSH negative control (Fig. 3A,B). This indicates that follicle growth can be maintained to some extent independently of FSH action until follicles can no longer survive, as was observed in conditions of 1 ng/ml FSH²⁴ or no FSH treatment by D8-D12 of culture (Fig. 3A,B, Fig. 2A,B). Follicles grown in the absence of FSH reached a maximum average diameter of 158.4±19.72 µm (range: 131.3-194.6 µm), demonstrating that cultured early secondary follicles transition from an FSH-independent to an FSH-dependent state before reaching 200 µm (Fig. 3B).

Although no difference in growth was observed on D4 between treatments (Fig. 3C), by D6 and through the remaining duration of culture, FSH²¹ resulted in a significant increase in growth compared with equivalent concentrations of FSH²⁴ (Fig. 3D-F); 10 ng/ml treatment with FSH²¹ resulted in a 1.56-, 1.28-, 1.18-fold increase in growth on D6, D8 and D12, respectively, compared with equivalent treatment with FSH²⁴. Between D6 and D12 of culture, 1 ng/ml FSH²¹ treatment resulted in an increase in size of 1.45- to 1.56-fold compared with the equivalent treatment of FSH²⁴. At 20 and 100 ng/ml concentrations, FSH²¹ and FSH²⁴ induced similar increases in growth, except for on D6 when FSH²¹-treated follicles were significantly larger (Fig. S1A,B). Because 1 ng/ml and 10 ng/ml concentrations were the minimum doses of FSH²¹ and FSH²⁴ that could maintain survival, respectively, we used these concentrations for further analyses.

Aromatase expression and consequently estradiol production by follicles is highly sensitive to FSH, and steroidogenic activity is maintained in the eIVFG system (Converse et al., 2023). Therefore, we assessed estradiol levels in conditioned media to determine whether the differences elicited by FSH glycoforms on follicle growth were also reflected in functional consequences on steroidogenesis. Treatment with 10 ng/ml FSH²¹ resulted in a significant increase in estradiol secretion compared with all other treatments between D8 and D12 of culture (Fig. 4). For the 10 ng/ml treatments, estradiol levels were 13.4- and 4.5-fold higher with FSH²¹ treatment compared with treatment with FSH²⁴ on D8 and D12, respectively.

To determine whether the FSH²¹-mediated increase in follicle growth and estradiol production translates into improved gamete quality, we assessed the ability of oocytes of in vitro-grown follicles to produce MII-arrested eggs in response to ex vivo ovulation cues. We only evaluated follicles grown in the presence of 10 ng/ml FSH²¹ and FSH²⁴ conditions owing to the relatively poor growth and survival in the 1 ng/ml FSH²⁴-treated follicles. FSH²¹ treatment resulted in significantly more follicles (67.9%) that were large enough (>180 µm) to be used for ex vivo ovulation compared with FSH²⁴ treatment (46.7%) (Fig. 5A). Comparison of the terminal diameters of mature follicles selected for induction of maturation indicated no significant difference in follicle size between glycoform treatments (Fig. 5B), demonstrating that the populations used to assess oocyte maturation and quality were similar in terms of stage. After ex vivo ovulation, meiotic stage and MII-spindle configuration were assessed by immunocytochemistry. The production of a mature egg arrested at metaphase of meiosis II was confirmed by the presence of an extruded polar body and a meiotic spindle. There was no significant difference in the number of oocytes that reached MII between glycoform treatments (Fig. 5C). Normal MII spindles were barrel-shaped, bipolar, and exhibited chromosomes aligned on the metaphase plate, whereas abnormal MII spindles had misaligned chromosomes and/or additional poles as demonstrated with tubulin connections to additional pericentrin foci (Fig. 5D). A greater proportion of MII eggs from FSH²¹-treated follicles exhibited normal MII spindle configurations (76.32%) compared with FSH²⁴-treated follicles (50%) (Fig. 5E). Of the abnormal oocytes from FSH²¹-treated follicles, 72.7% had spindle abnormalities, 18.2% had misaligned chromosomes and 9.1% had both. Abnormalities in oocytes from FSH²⁴-treated follicles consisted of 62.5% spindle abnormalities, 6.3% chromosome misalignment and 31.2% with both. Between glycoform treatments, no difference in spindle length or width was observed in MII eggs with normal spindle configurations (Fig. 5F,G). Collectively, these studies indicate that although both FSH glycoforms support follicles that generate meiotically competent gametes, follicles grown in FSH²⁴ produce eggs with chromosome and spindle defects that will compromise their quality and developmental potential.

To investigate the mechanism by which FSH²¹ confers a beneficial impact on follicle growth, function and gamete quality, we first examined FSH-mediated signaling and transcriptomic profiles of follicles treated with FSH²¹ and FSH²⁴. Analysis of CREB phosphorylation (activation) after 15 min treatment with 1 or 10 ng/ml concentrations of each glycoform indicated a significant effect of FSH concentration on CREB activity, with 1 ng/ml treatments resulting in fold increases in phospho-CREB of 4.7±2.0 and 1.6±1.3 for FSH²¹ and FSH²⁴ compared with control, respectively (Fig. S2A). Treatments with 10 ng/ml resulted in higher phospho-CREB levels (24.4±15.9 fold and 15.9±10.6 fold compared with control for FSH²¹ and FSH²⁴, respectively), than that induced by 1 ng/ml treatments, but phospho-CREB levels were not different between equivalent glycoform concentrations. Both concentrations of each glycoform increased PKA substrate phosphorylation above control to similar levels after 12 h (Fig. S2B). These analyses indicate that both glycoforms elicit FSH-responsive signaling cascades, but members of the PKA-CREB signaling pathway are not differentially modulated by the different glycoforms. RNA-sequencing (RNA-seq) analysis of follicles treated with 10 ng/ml FSH²¹ or FSH²⁴ for 24 h identified 71 differentially expressed genes (DEGs; log2FC≥1, P<0.05) (Fig. 6A), but only 38 were protein-coding (Fig. 6B). However, compared with ‘no FSH’ control, FSH²¹ and FSH²⁴ treatments resulted in 393 and 270 DEGs, respectively, with 221 DEGs shared between glycoform treatments. This indicates that, at this time point, FSH glycoforms exhibit similar effects on transcription.

Owing to the minimal transcriptional changes observed at 24 h, we next investigated whether FSH glycoforms differentially regulate cell–cell interactions and bidirectional communication within the follicle. Transzonal projections (TZPs) are granulosa-derived, cytoplasmic projections that establish oocyte–GC communication through gap junctions consisting of connexin 37 (encoded by Gja4) (Clarke, 2018). Additionally, connexin 43 (encoded by Gja1) establishes gap junctions between GCs within ovarian follicles. TZP number and connexin expression positively correlate with oocyte and follicle size (El-Hayek et al., 2018; Teilmann, 2005), and FSH is a known modulator of connexin expression and TZPs in murine follicles (Carabatsos et al., 2000; El-Hayek and Clarke, 2015; Sommersberg et al., 2000; Wang et al., 2013). Therefore, we reasoned that FSH glycoforms would have differential effects on the ability of follicles to establish TZPs and gap junctions, which could contribute to differences in follicle growth and oocyte quality. To examine this, early secondary follicles were incubated for 24 h with 1 or 10 ng/ml of each FSH glycoform and then stained for actin, as the majority of TZPs are actin-based (El-Hayek et al., 2018), the TZP-associated protein MYO10 (Granados-Aparici et al., 2022), or connexin 43, which is the principle gap junction protein in GC-GC gap junctions (Ackert et al., 2001). Actin staining in the area of the zona pellucida was significantly higher in 10 ng/ml FSH²¹-treated follicles compared with all other treatments, with a 2.4-fold increase compared with control and a 1.6-fold increase compared with the equivalent treatment with FSH²⁴ (Fig. 7A,D). Treatment with 10 ng/ml FSH²¹ increased MYO10 foci by 1.9-fold compared with the control and 5.1-fold compared with the 10 ng/ml FSH²⁴ treatment (Fig. 7B,E). Similarly, 10 ng/ml FSH²¹ resulted in a 1.6-fold increase in connexin 43 foci compared with control and a 4.1-fold increase compared with the equivalent FSH²⁴ treatment (Fig. 7C,F). This indicates that 10 ng/ml FSH²¹ resulted in higher levels of TZPs and connexin 43 plaques between GCs within 24 h of treatment relative to the equivalent concentrations of FSH²⁴. Of note, there was no difference in the size of follicles between treatments at this time point (Fig. 7G). Thus, the increase in cell-to-cell association in the 10 ng/ml FSH²¹-treated follicles was not due to the differences in terminal follicle sizes. Overall, these data demonstrate that FSH²¹ stimulates early oocyte–GC and GC–GC association, which precedes follicle growth but likely confers the subsequent growth advantage owing to enhanced cellular communication. The expression of genes known to be correlated with follicle cell-to-cell communication, including Gja4 (connexin 37; Gittens and Kidder, 2005; Simon et al., 1997), Gja1 (connexin 43; Ackert et al., 2001; Gittens and Kidder, 2005), Myo10 (Crozet et al., 2023; Granados-Aparici et al., 2022), Ctnnb1 (Wang et al., 2013), Cdh1 (El-Hayek and Clarke, 2015), Cdh2 (El-Hayek and Clarke, 2015), Daam1 (El-Hayek et al., 2018) and Fscn1 (El-Hayek et al., 2018), were not differentially regulated between 10 ng/ml FSH²¹ and FSH²⁴ treatments (Fig. 7H). This suggests that the FSH²¹-mediated cell-to-cell communication is independent of transcription within the time frame examined.

To determine whether the increased interaction between cells within follicles results in increased follicle growth, we examined 5-ethynyl-2′-deoxyuridine (EdU) staining of follicle GCs after 24 h treatment with 10 ng/ml of each glycoform as an early indicator of cell proliferation. Follicles treated with FSH²¹ exhibited a 2.4-fold increase in the number of EdU-labeled GCs compared with control (Fig. 8A). FSH²⁴ treatment also increased (1.6-fold) the number of EdU-labeled GCs compared with control, but to a significantly lesser extent than FSH²¹ treatment, which was 1.5-fold higher. Given that 10 ng/ml FSH²¹ treatment results in an increase in TZPs, GC-specific gap junctions, and increased levels of GC proliferation compared with FSH²⁴, we next sought to determine whether cell-to-cell communication directly modulates the proliferative ability of FSH²¹. Co-treatment of follicles with 10 ng/ml FSH²¹ and the pan gap junction inhibitor carbenoxolone (CBX, 100 µm) resulted in abrogation of the positive effect of FSH²¹ on GC proliferation, resulting in EdU labeling similar to control levels (Fig. 8B). Treatment with the inhibitor alone showed no difference in the proportion of EdU-labeled GCs compared with control. This indicates that FSH²¹ mediates GC proliferation through a gap junction-dependent mechanism. To determine whether the gap junction-dependent early proliferation induced by FSH²¹ at 24 h influences long-term follicle growth trajectories and oocyte outcomes, co-treatment with FSH²¹ and CBX was performed for 24 h. Follicles were then cultured with only FSH²¹ through the remaining duration of long-term culture. Of note, transient CBX treatment did not influence follicle survival (92±6.9% survival on D10, compared with 95±6.6% survival for FSH²¹ control), indicating that short-term CBX treatment is not toxic to follicles. However, on D10 of culture, follicles transiently co-treated with CBX were significantly smaller than those treated with FSH²¹ alone, increasing in size from 105.2±9.6 to 182±35.6 µm compared with 106.3±8.6 to 205.1±37.2 µm for FSH²¹ control (Fig. 9A,B). Normalized estradiol levels were also significantly lower in D9 culture media from follicles transiently treated with CBX (Fig. 9C). Lastly, transient CBX treatment resulted in an increased proportion of MII eggs that exhibited spindle and/or chromosome alignment abnormalities (Fig. 9D). These findings demonstrate that the positive effects of FSH²¹ on folliculogenesis and oocyte development are dependent on early establishment of cell-to-cell communication.

We demonstrate that hypoglycosylated FSH²¹ enhances folliculogenesis and improves gamete quality compared with fully glycosylated FSH²⁴. As summarized in Fig. 10, mechanistically, FSH²¹ drives early establishment of gap junction-mediated cell-to-cell interactions in the secondary follicle, which enables increased GC proliferation as well as follicular growth and function. The resulting gametes, in turn, exhibit improved quality as evidenced by a higher percentage of mature eggs with normal meiotic spindle and chromosome configurations. These findings extend the known differences in bioactivities of FSH glycoforms to the context of early follicle development and provide evidence that the age-dependent shift in FSH glycoform abundance likely contributes to decreased oocyte quality.

In prior work, the direct effect of FSH glycoforms on ovarian follicles has only been assessed in shorter term assays with pre-antral follicles, with a focus on follicle-specific outcomes. We previously performed short-term analysis of FSH²¹ and FSH²⁴ (100 ng/ml) induction of FSH-responsive signaling and gene expression in early secondary follicles (Simon et al., 2019). Similar to the results in the current study, PKA substrate phosphorylation levels were similar between glycoform treatments at 1 and 18-20 h. Furthermore, the majority of target genes assessed showed similar responsiveness between glycoform treatments at 1 and 18-20 h. Our non-biased RNA-seq analysis confirmed that FSH²¹ and FSH²⁴ have similar effects on gene expression changes in the short term. Recent work by Johnson et al. (2022) also examined the effect of 10 ng/ml concentrations on pre-antral follicle growth, survival and expression of FSH-responsive and apoptosis-associated genes. This study found minimal glycoform-specific effects on FSH-responsive genes at 24 h of treatment, but by 48 h Fshr, Cyp19a1, Amh, Bmp15 and Gdf9 showed upregulation with FSH²¹ treatment and downregulated with FSH²⁴ treatment. Thus, glycoform-specific transcriptomic differences may become more consequential and likely to provide greater insights at later time points of FSH treatment/signaling. These findings indicate that in terms of early FSH-induced PKA-CREB signaling and transcriptomic regulation, the difference between glycoform treatment is minimal and indicates that other molecular or cellular changes may be dictating differential folliculogenesis outcomes.

FSH promotes gap junction formation between GCs from all follicle classes (60-450 µm) (Burghardt and Matheson, 1982) and stimulates expression and plaque formation of connexins 43 (GC specific) and 37 (oocyte specific) in murine GC lines and pre-antral follicles (El-Hayek and Clarke, 2015; Sommersberg et al., 2000; Wang et al., 2013). FSH also modulates TZPs in vitro and in vivo (El-Hayek and Clarke, 2015). We demonstrate that FSH²¹ is more effective at promoting TZP formation as well as connexin 43 plaque formation between GCs, and that such connections are essential for the early effect of FSH²¹ on GC proliferation. We demonstrate that the positive effects of FSH on cell-to-cell communication modulate the well-known role of FSH in mediating GC proliferation (Robker and Richards, 1998), with bidirectional communication likely driving the tight positive association between gap junction protein expression and TZP number with ovarian follicle and oocyte size (El-Hayek et al., 2018; Teilmann, 2005). The exact mechanism by which FSH²¹ increases early establishment of this cell-to-cell communication requires further investigation but appears to be independent of transcription based on our RNA-seq large-scale gene expression data. Future work examining the mechanism responsible for the increase in gap junctions and TZPs in FSH²¹-treated follicles will set the stage for the potential experimental modulation of these early crucial events to further interrogate their role in FSH glycoform-specific effects.

Gap junctions are essential for proper folliculogenesis as ovarian cell-specific knockout models of connexins 43 (43^+/+oocyte/43^–/–GC) and 37 (37^–/–oocyte/37^+/+GC) exhibit abrogated follicle formation and growth and produce oocytes that lack meiotic competence (Ackert et al., 2001; Gittens and Kidder, 2005; Simon et al., 1997). Furthermore, disruption of oocyte–GC communication results in abrogated oocyte growth and disrupted nuclear and cytoplasmic maturation competence (Carabatsos et al., 2000; Eppig, 1979). Such cell-to-cell communication within the follicle mediates metabolic cooperativity between the oocyte and somatic cells to support glycolysis, amino acid uptake and cholesterol biosynthesis in the oocyte (reviewed by Doherty et al., 2022). Our work indicates that FSH²¹ is more efficient at promoting the establishment of cell-to-cell interactions within the first 24 h of FSH exposure compared with FSH²⁴ treatment. Although time points beyond 24 h were not examined in terms of cell-to-cell association, we predict that increased bidirectional communication is ultimately established in FSH²⁴-treated follicles given that follicle growth is able to progress. The beneficial effect of FSH²¹ occurs early during folliculogenesis because its absence during the initial 24 h of culture recapitulates phenotypes of FSH²⁴-treated follicles, including decreased folliculogenesis and oocyte quality parameters. Thus, the FSH²¹-induced increase in cell-to-cell communication in early secondary follicles is essential for folliculogenesis and establishment of a high-quality gamete later in development. Of note, FSH²¹-treated follicles also produced significantly more estradiol compared with those treated with FSH²⁴. Estradiol is also a known promoter of granulosa proliferation, and follicle growth is modulated by both FSH and estradiol at different time points in folliculogenesis (Rao et al., 1978). Determining the mechanism for the increase in estradiol secretion in FSH²¹- but not FSH²⁴-treated follicles as well as deciphering the independent contributions of FSH glycoforms and estradiol to later stages of follicle growth are important areas of future investigation.

The age-associated shift in FSH glycoform abundance occurs coordinately with the age-dependent decline in gamete quality and may underlie this phenomenon. In support of this, our current work demonstrates that hypoglycosylated FSH²¹, which is more physiologically available during the peak of reproductive potential in females (Bousfield et al., 2014a), exhibits an enhanced capacity to promote the development of mature MII-arrested eggs with normal spindle configurations and chromosome alignment compared with FSH²⁴. Moreover, FSH²⁴-treated follicles produce gametes that exhibit phenotypes that are often observed with physiological reproductive aging, such as altered spindle morphology and aneuploidy (Pan et al., 2008; Shomper et al., 2014). Differences in FSH glycoform modulation of cell-to-cell communication may contribute to these phenotypes, as impaired bidirectional communication can disrupt oocyte growth and developmental competence (Carabatsos et al., 2000; Eppig, 1979). Of interest, follicles from mice of advanced reproductive age have reduced TZPs and decreased expression of filopodia-associated genes (El-Hayek and Clarke, 2015). Our future work will test whether age-specific FSH glycoforms exhibit differences in bioactivity on follicles obtained from reproductively young and old adult mice.

A proportion of follicles grown in the presence of FSH²⁴ still produced normal mature MII-arrested eggs, and there was no difference in follicle survival between 10 ng/ml glycoform conditions. These findings indicate that FSH²⁴ is not detrimental to folliculogenesis, which contrasts with data from a previous study in which FSH²⁴ treatment induced apoptosis and follicle death (Johnson et al., 2022). The physiological relevance of these findings is unclear because FSH²⁴-induced cell death would result in complete abrogation of fertility in the perimenopausal period when FSH²⁴ is the most abundant form. Such an activity would also be expected to be detrimental in assisted reproductive technologies wherein the majority of commercially available recombinant FSH preparations used for follicle stimulation predominantly contain FSH²⁴ (Bousfield et al., 2014a; Butnev et al., 2015). It is thus clear that FSH²⁴ is less potent compared with FSH²¹ in coordinating the cellular and molecular events that are beneficial to follicle and oocyte development. This has important implications for clinical assisted reproductive technology as well as follicle culture methods, which use recombinant FSH sources typically dominated by FSH²⁴ (Bousfield et al., 2014a; Butnev et al., 2015). Shifting the composition of these preparations towards a higher fraction of FSH²¹ may represent a translational opportunity to improve folliculogenesis and oogenesis outcomes.

Follicles used in this experiment were isolated from prepubertal CD-1 mice. Dams with pups were obtained from Envigo (Indianapolis, IN, USA), and prepubertal mice were used for experiments between postnatal days 12 and 16 to maximize the yield of a uniform population of early secondary stage follicles. Animals were maintained in accordance with the National Institutes of Health's guidelines and housed in Northwestern University's Center for Comparative Medicine barrier facility under constant light (12 h light/12 h dark), humidity, and temperature control, and offered food and water ad libitum. All animal experiments were approved by Northwestern University's Institutional Animal Care and Use Committee.

Early secondary follicles (95-125 µm in diameter) were isolated from ovaries and encapsulated in 0.5% alginate as previously described (Converse et al., 2023). All culture media and supplements were from Gibco unless otherwise specified. Briefly, follicles were mechanically isolated using insulin syringes in L15 media supplemented with 1% fetal bovine serum and 0.5% penicillin-streptomycin, allowed to recover for a minimum of 1 h in α-MEM-Glutamax at 37°C in a humidified atmosphere of 5% CO₂, then encapsulated in 0.5% alginate (Sigma-Aldrich) in groups of ten. Only follicles with healthy morphology (well-defined oocyte plasma membrane and distinct GC layers) were encapsulated. Alginate beads containing follicles were plated in polystyrene 96-well plates in 100 µl of growth media [α-MEM-Glutamax containing 3 mg/ml bovine serum albumin (MP Biomedicals), 1 mg/ml fetuin (Sigma-Aldrich) and 0.1% insulin-transferrin-selenium (Thermo Fisher Scientific)]. Depending on the experiment, the growth media was supplemented with hypoglycosylated FSH²¹ or fully glycosylated FSH²⁴ (1, 10, 20, 100 ng/ml). FSH glycoforms were purified from rat pituitary GH₃ clones co-expressing hypo- and fully glycosylated FSH glycoforms following previously described methods (Bousfield et al., 2014b; Butnev et al., 2015; Jiang et al., 2015). The in vivo bioactivity of these recombinant hFSH glycoforms was previously tested using Fshb null female mice (Wang et al., 2016).

Follicles were maintained in culture at 37°C, under humidified conditions of 5% CO₂ in air for up to 12 days, with half media changes performed every other day. For FSH²¹ and CBX (pan gap-junction inhibitor, 100 µm) co-treatment experiments, follicles were treated in ultra-low adherent 96-well plates for 24 h, washed three times in growth media (without FSH), then incubated in growth media (without FSH) for 1 h prior to encapsulation in alginate and long-term culture. Follicles were imaged using a 10× objective on an Evos FL Auto imaging system (Thermo Fisher Scientific) on D0 or D1 (co-treatment experiments) and every other day throughout the duration of culture. Individual follicles were tracked by monitoring their relative positions within the alginate bead. Follicle growth was analyzed by averaging two perpendicular diameter measurements for each follicle using ImageJ software (NIH, Bethesda, MD, USA) and plotting these values across the culture period. Follicle survival was determined by morphological appearance, with follicles exhibiting deterioration (dark GCs or unhealthy oocyte) or absence of growth over four consecutive culture days being categorized as dead.

RNA was isolated from pooled follicles, ovaries (from control or Fshr^−/− mice), or pituitaries (from control mice) using RNeasy Mini columns (QIAGEN), DNaseI (Thermo Fisher Scientific) treated, and cDNAs were synthesized using SuperScript-III reverse transcriptase kit as described (Liu et al., 2022). Each PCR reaction was performed in 10 μl of reaction volume containing 2 μl of cDNA diluted at 1:40, 0.05 µM each of primer/probe combos, and 5 μl of 2× PrimeTime Gene Expression Master Mix (Integrated DNA Technologies) using QuantStudio 6 Flex Real-Time PCR system. The relative standard curve method was used for gene expression quantification as described (Liu et al., 2022). For each primer, a series of dilution of standard cDNA at 1:5, 1:10 and 1:50 were assigned the quantity as 2000, 1000 and 200. Relative mRNA levels of Fshr (exons 1-3 and exons 9-10) normalized to Ppil1 were obtained and the ratios are presented.

RNA-sequencing libraries were generated by the University of Nebraska Medical Center Sequencing Core beginning with 50 ng of total RNA from each sample using the NuGEN Universal Plus mRNA-seq library kit from TECAN following the manufacturer's recommended procedure as described (McDonald et al., 2023). Sequencing data were demultiplexed, and FASTQ files were generated using bcl2fastq2 software (Illumina, version 2.20.0). Sequencing quality control was performed using FastQC tool developed by Babraham Institute and Bioinformatics analysis was performed as described (McDonald et al., 2023). A gene was characterized as differentially expressed if the false discovery rate cutoff using the Benjamini–Hochberg procedure for multiple testing correction was ≤0.05 and the absolute fold change was above 1.0.

For analysis of FSH-induced CREB and PKA activation, follicles were plated without encapsulation on ultra-low adherent 96-well plates. Follicles were first cultured in FSH-free growth media for a minimum of 6 h for PKA activity analysis and 24 h for CREB activity analysis to ensure that protein and substrate phosphorylation was only due to exogenous FSH treatment rather than endogenous FSH exposure. After starvation, follicles were placed in growth media containing various concentrations of FSH glycoforms for 15 min (CREB activation) or 12 h (PKA activation). At the end of treatment, follicles were rinsed through PBS containing 0.3% (w/v) polyvinylpyrrolidone, snap-frozen on dry ice, and stored at −80°C. To analyze PKA and CREB activity, western blots were performed with the follicle protein extracts. All primary and secondary antibodies used are listed in Table S1. Electrophoresis was performed on sets of ten follicles loaded on 4-15% premade SDS-polyacrylamide gels (Bio-Rad Laboratories) under reducing conditions. The separated proteins were transferred to a PVDF membrane, blocked with 5% bovine serum albumin (BSA; Sigma-Aldrich) for 2 h, and incubated in primary antibodies (in 5% BSA; anti-phospho-PKA substrate or anti-phospho-CREB) overnight at 4°C. Membranes were then washed in Tris-buffered saline containing 1% Tween-20 (Sigma-Aldrich) (TBS-T), incubated in conjugated secondary antibodies (ECL anti-rabbit, HRP-linked) for 1 h at room temperature, then washed again with TBS-T. The protein-antibody complexes were detected using Amersham ECL Plus chemiluminescence reagents (GE Healthcare). Blots were then stripped using Restore Western Blot Stripping Buffer (Thermo Fisher Scientific), re-blocked, and probed with antibodies against total-CREB or α-tubulin for normalization of protein loading. Protein bands were visualized using chemiluminescence film and band densitometry was assessed using ImageJ software.

17β-Estradiol levels were assessed in conditioned media from follicle culture using a commercial immunoassay (Caymen Chemicals) with a detection sensitivity of 20 pg/ml following the manufacturer's protocol. Media was diluted 1:5-1:10 in assay buffer to ensure readings were obtained between 20-80% of maximum binding as recommended by the manufacturer. Data are reported as estradiol ng/ml or normalized estradiol, as in the case when high variability in raw values was indicated between biological experimental replicates.

To assess the meiotic competence of oocytes from follicles grown in culture, follicles were removed from alginate on D10 of culture by incubation with 10 IU/ml alginate lyase (Sigma-Aldrich) for 15-30 min at 37°C, followed by rinsing in L15 media. Follicles ≥180 µm in diameter were transferred to maturation media [α-MEM-Glutamax containing 10% fetal bovine serum, 10 ng/ml mouse epidermal growth factor (EGF; BD Biosciences), 1.5 IU/ml hCG (Sigma-Aldrich) and 10 mIU/ml FSH (Organon)] for 16 h at 37°C in a humidified atmosphere of 5% CO₂ (Skory et al., 2015; Xu et al., 2006). Oocytes were then recovered from ovulated follicles and processed for whole-mount immunocytochemistry as described below.

Whole-mount immunocytochemistry (ICC) was performed on oocytes for meiotic spindle analysis and on early secondary follicles to evaluate TZPs and connexin 43 expression. All antibody suppliers and concentrations are listed in Table S1. For spindle analysis, antibodies against α-tubulin and pericentrin were utilized. The actin cytoskeleton was visualized using phalloidin. Oocytes were fixed in 3.8% paraformaldehyde containing 0.1% Triton X-100 (Alfa Aesar) at 37°C for 20 min; follicles were fixed under the same conditions but without 0.1% Triton X-100. After fixation, oocytes or follicles were washed three times in PBS (Thermo Fisher Scientific) and stored at 4°C until processed for ICC. Whole-mount samples were permeabilized in PBS containing 1% BSA and 0.1% Triton X-100 for 15 min at room temperature, rinsed in blocking buffer (PBS containing 1% BSA and 0.1% Tween-20) three times (5 min each), then incubated in primary antibody (diluted in blocking buffer) overnight at 4°C. Samples were washed three times with blocking buffer, incubated with secondary antibodies or fluorophore-conjugated primary antibodies for 2 h at room temperature protected from light, washed three times with blocking buffer, then mounted onto microscope slides with Vectashield Plus Antifade Mounting Medium containing 4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories). Slides were stored at 4°C. Whole-mount samples were imaged with a Leica TCS Sp5 confocal system using 40× or 63× objectives (Leica Microsystems).

For each oocyte, optical z-sections were acquired at 1.0 µm intervals to span the region of the entire MII spindle, and image analysis was performed using ImageJ. z-sections were merged into a projection and meiotic spindles were scored as normal or abnormal. Normal spindles were barrel-shaped and bipolar as determined by pericentrin localization to each pole and exhibited chromosome alignment along the metaphase plate. Spindle length was determined as the distance between the two poles using the Pythagorean theorem, where a² is the two-dimensional length between spindle poles on the projection image and b² is the z-range between poles. The widest horizontal plane of each spindle was used for spindle width analysis.

Follicles were encapsulated in sets of ten and cultured in growth media with various concentrations of the FSH glycoforms for 24 h, after which follicles were removed from alginate beads, washed and fixed as described above. Follicles were stained with F-actin using phalloidin or with antibodies towards the TZP-associated protein MYO10 (Crozet et al., 2023; Granados-Aparici et al., 2022) or connexin 43. Optical sections of the equilateral plane of the oocyte were acquired for all follicles and used for analysis. To compare signal intensities, all follicles within an experiment underwent ICC processing simultaneously and with the same antibody or staining solutions, and imaging was performed with constant laser power and gain settings across all experimental cohorts. Actin staining intensity in the region of the zona pellucida, indicative of TZP density, was determined using previously described methods (El-Hayek and Clarke, 2015). Briefly, concentric circles were drawn using ImageJ to encompass the region of the zona pellucida, taking care to exclude the actin-stained plasma membranes of the oocyte and GCs. The mean gray value of the area between the circles was determined, and background intensity calculated as the mean gray value from a region of interest drawn in an actin-free region of the follicle (background) was subtracted. For MYO10 and connexin 43 analyses, images were acquired as described above, and the number of positive foci within single optical planes was determined using ImageJ. Four optical planes were assessed for each follicle, and the average number of foci per plane was used for analysis. Fluorescent signal thresholding, image processing and inclusion criteria were maintained between all treatments.

A Click-iT EdU imaging kit (Thermo Fisher Scientific) was used to label GCs in S phase following the manufacturer's instructions. Briefly, after glycoform treatment, follicles were moved to L15 media (without FSH) containing 10 µM EdU and incubated for 30 min at 37°C. Follicles were then fixed in 3.8% paraformaldehyde for 30 min at 37°C, washed three times with 3% BSA in PBS, permeabilized with 0.5% Triton X-100 in PBS for 20 min at room temperature, then washed again in 3% BSA before following the EdU labeling protocol. Follicles were subsequently mounted in Vectashield Plus Antifade Mounting Medium containing DAPI.

Statistical significance was determined by one-way ANOVA with a post-hoc Bonferroni multiple comparison test or χ² test, with P<0.05 considered significant. One-way ANOVA was used to analyze experimental results with multiple glycoform treatments, and χ² tests were used on categorical data. All data are expressed as mean±s.e.m. using Prism 9.4 software (GraphPad Software). Only significant findings are described in the results.

We acknowledge Prianka H. Hashim and Kathryn Trotter for their help with follicle isolation for RNA-seq experiments, and James Eudy and the Nebraska University Medical Center DNA sequencing core for help with RNA-seq experiments. The University of Nebraska DNA Sequencing Core was supported in part by the National Institute for General Medical Science (NIGMS) INBRE - P20GM103427-19 grant as well as The Fred & Pamela Buffett Cancer Center Support Grant - P30 CA036727. The Bioinformatics and Systems Biology Core (BSBC) received partial support from NIH awards (P20GM103427, P30CA036727, U54GM115458).

The peer review history is available online at https://journals.biologists.com/dev/lookup/doi/10.1242/dev.202170.reviewer-comments.pdf